Methods of therapy for b-cell malignancies using antagonist anti-cd40 antibodies

ABSTRACT

Methods of therapy for B-cell malignancies are provided. The methods comprise administering a therapeutically effective amount of an antagonist anti-CD40 antibody or antigen-binding fragment thereof to a patient in need thereof. The antagonist anti-CD40 antibody or antigen-binding fragment thereof is free of significant agonist activity when the antibody binds a CD40 antigen on a normal human B cell, exhibits antagonist activity when the antibody binds a CD40 antigen on a malignant human B cell, and can exhibit antagonist activity when the antibody binds a CD40 antigen on a normal human B cell. Antagonist activity of the anti-CD40 antibody or antigen-binding fragment thereof beneficially inhibits proliferation and/or differentiation of malignant human B cells.

FIELD OF THE INVENTION

[0001] The present invention is directed to methods of therapy fordiseases characterized by malignant B cells and tumors of B-cell originusing antagonist anti-CD40 antibodies or antigen-binding fragmentsthereof

BACKGROUND OF THE INVENTION

[0002] B cells play an important role during the normal in vivo immuneresponse. A foreign antigen will bind to surface immunoglobulins onspecific B cells, triggering a chain of events including endocytosis,processing, presentation of processed peptides on MHC-class IImolecules, and up-regulation of the B7 antigen on the B-cell surface. Aspecific T-cell then binds to the B cell via T-cell receptor (TCR)recognition of the processed antigen presented on the MHC-class IImolecule. Stimulation through the TCR activates the T cell and initiatesT-cell cytokine production. A second signal that further activates the Tcell is an interaction between the CD28 antigen on T cells and the B7antigen on B cells. When the above-mentioned signals are received, theCD40 ligand, which is not expressed on resting human T cells, isup-regulated on the T-cell surface. Binding of the CD40 ligand to theCD40 antigen on the B-cell surface stimulates the B cell, causing the Bcell to mature into a plasma cell secreting high levels of solubleimmunoglobulin.

[0003] CD40 is a cell-surface antigen present on the surface of bothnormal and neoplastic human B cells, dendritic cells, monocytic andepithelial cells, some epithelial carcinomas, and on antigen presentingcells (APCs). CD40 expression on APCs plays an important co-stimulatoryrole in the activation of both T helper and cytotoxic T lymphocytes.CD40 receptors are also found on eosinophils, synovial membranes inrheumatoid arthritis, activated platelets, inflamed vascular endothelialcells, dermal fibroblasts, and other non-lymphoid cell types. The CD40receptor is expressed on activated T cells, activated platelets, andinflamed vascular smooth muscle cells. CD40 is also expressed at lowlevels on vascular endothelial cells and is up-regulated in areas oflocal inflammation.

[0004] Human CD40 is a peptide of 277 amino acids having a predictedmolecular weight of 30,600, with a 19 amino acid secretory signalpeptide comprising predominantly hydrophobic amino acids. The CD40receptor exists in a highly modified glycoprotein state on the cellsurface and migrates in sodium dodecyl sulfate (SDS)-polyacrylamide gelsas an approximately 50 kDa polypeptide.

[0005] The CD40 antigen is known to be related to the human nerve growthfactor (NGF) receptor, tumor necrosis factor-α (TNF-α) receptor, andFas, suggesting that CD40 is a receptor for a ligand with importantfunctions in B-cell activation. During B-cell differentiation, themolecule is first expressed on pre-B cells and then disappears from thecell surface when the B cell becomes a plasma cell. The CD40cell-surface antigen plays an important role in B-cell proliferation anddifferentiation.

[0006] Binding of its ligand (termed CD40L or CD154) to the CD40receptor stimulates B-cell proliferation and differentiation, antibodyproduction, isotype switching, and B-cell memory generation. The humanand murine CD40L (CD40 receptor) genes have been cloned (Spriggs et al.(1992) J. Exp. Med. 176:1543; Armitage et al. (1992) Nature 357:80; andU.S. Pat. No. 6,264,951). Engagement of CD40 receptors by the CD40ligand on APCs, such as macrophages and dendritic cells, up-regulatescell-surface expression of MHC Class II and CD80/86, and induces thesecretion of pro-inflammatory cytokines such as IL-8, IL-12, and TNF,all of which increase the potency of antigen presentation to T cells.

[0007] All B cells express common cell surface markers, including CD40.Transformed cells from patients with low- and high-grade B-celllymphomas, B-cell acute lymphoblastic leukemia, multiple myeloma,chronic lymphocytic leukemia, and Hodgkin's disease express CD40. CD40expression is also detected in two-thirds of acute myeloblastic leukemiacases and 50% of AIDS-related lymphomas. Further, malignant B cells fromseveral tumors of B-cell lineage express a high degree of CD40 andappear to depend on CD40 signaling for survival and proliferation.

[0008] Additionally, immunoblastic B-cell lymphomas frequently arise inimmunocompromised individuals such as allograft recipients and othersreceiving long-term immunosuppressive therapy, AIDS patients, andpatients with primary immunodeficiency syndromes such as X-linkedlymphoproliferative syndrome or Wiscott-Aldrich syndrome (Thomas et al.(1991) Adv. Cancer Res. 57:329; Straus et al. (1993) Ann. Intern. Med.118:45). These tumors appear to arise as a result of impaired T-cellcontrol of latent Epstein-Barr virus (EBV) infection. Similar lymphomasof human origin can be induced in mice with severe combinedimmunodeficiency syndrome (SCID) by inoculation of peripheral bloodlymphocytes (PBL) from healthy, EBV-positive individuals (Mosier et al.(1988) Nature 335:256; Rowe et al (1991) J. Exp. Med. 173:147).

[0009] The pathogenesis of low-grade B-lineage malignancies, includingnon-Hodgkin's lymphoma and chronic lymphocytic leukemia, is stronglyaffected by the imbalance of the growth/survival signal by CD40 and acrippled death signal by Fas. Studies in low-grade non-Hodgkin'slymphoma suggest that the disease is the result of an accumulation oflymphomatous cells due to reduction in Fas-mediated apoptosis and anincrease in the survival signal through CD40. CD40 provides a survivalsignal for lymphoma cells from non-Hodgkin's B-lymphoma patients andstimulates their growth in vitro (Romano et al (2000) Leuk Lymphoma36:255-262; Furman et al. (2000) J. Immunol. 164:2200-2206; Kitada etal. (1999) Br. J. Haematol. 106:995-1004; Romano et al. (1998) Blood92:990-995; Jacob et al. (1998) Leuk Res. 22:379-382; Wang et al. (1997)Br. J. Haematol. 97:409-417; Planken et al. (1996) Leukemia 10:488-493;and Greiner et al. (1997) Am J. Pathol. 150:1583-1593).

[0010] Approximately 85% of non-Hodgkin's lymphomas, a diverse group ofmalignancies, are of B-cell origin. The non-Hodgkin's lymphomasoriginate from components of the spleen, thymus, and lymph nodes. In theWorking Formulation classification scheme, these lymphomas been dividedinto low-, intermediate-, and high-grade categories by virtue of theirnatural histories (see “The Non-Hodgkin's Lymphoma PathologicClassification Project,” Cancer 49(1982):2112-2135). The low-grade orfavorable lymphomas are indolent, with a median survival of 5 to 10years (Horning and Rosenberg (1984) N. Engl. J. Med. 311:1471-1475).Although chemotherapy can induce remissions in the majority of indolentlymphomas, cures are rare, and most patients eventually relapse,requiring further therapy. The intermediate- and high-grade lymphomasare more aggressive tumors, but they have a greater chance for cure withchemotherapy. However, significant numbers of these patients will stillrelapse and require further treatment to induce remissions. Furthermore,patients undergoing chemotherapy can experience toxicity effects.Therefore, there is a need for new therapies for treating diseases ofmalignant B cells.

SUMMARY OF THE INVENTION

[0011] Methods for treating a patient with a disease comprisingmalignant B cells, including lymphomas such as, non-Hodgkin's lymphomas(high-grade lymphomas, intermediate-grade lymphomas, and low-gradelymphomas), Hodgkin's disease, acute lymphoblastic leukemias, myelomas,chronic lymphocytic leukemias, and myeloblastic leukemias are provided.The method comprises treating the patient with anti-CD40 antibodies orantigen-binding fragments thereof that are free of significant agonistactivity when bound to a CD40 antigen on a normal human B cells and thatexhibit antagonist activity when bound to a CD40 antigen on a malignanthuman B cell. Monoclonal antibodies and antigen-binding fragmentsthereof that are suitable for use in the methods of the inventionexhibit the following characteristics: 1) are capable of specificallybinding to a human CD40 antigen expressed on the surface of a humancell; 2) are free of significant agonist activity when bound to a CD40antigen on a normal human B cell; and, 3) exhibit antagonist activitywhen bound to a CD40 antigen on a malignant human B cell. In someembodiments, the anti-CD40 antibody or fragment thereof also exhibitsantagonist activity when bound to CD40 antigen on normal human B cells.The monoclonal antibodies have a strong affinity for CD40 and arecharacterized by a dissociation constant (K_(d)) of at least 10⁻⁵ M,preferably at least about 10⁻⁸M to about 10⁻²⁰ M, more preferably atleast about 5×10⁻⁹ to about 10⁻¹⁶ M. Suitable monoclonal antibodies havehuman constant regions; preferably they also have wholly or partiallyhumanized framework regions; and most preferably are fully humanantibodies or antigen-binding fragments thereof Examples of suchmonoclonal antibodies are the antibody designated herein as 15B8, themonoclonal antibody produced by the hybridoma cell line designated 15B8,a monoclonal antibody comprising an amino acid sequence selected fromthe group consisting of SEQ ID NO:2 and SEQ ID NO:4; a monoclonalantibody comprising an amino acid sequence encoded by a nucleic acidmolecule comprising a nucleotide sequence selected from the groupconsisting of SEQ ID NO:1 and SEQ ID NO:3; and antigen-binding fragmentsof these monoclonal antibodies that retain the capability ofspecifically binding to human CD40.

[0012] In one embodiment of the invention, the therapy comprisesadministering to a patient a therapeutically effective dose of apharmaceutical composition comprising suitable anti-CD40 antibodies orantigen-binding fragments thereof. A therapeutically effective dose ofthe anti-CD40 antibody or fragment thereof is in the range from about0.01 mg/kg to about 40 mg/kg, from about 0.01 mg/kg to about 30 mg/kg,from about 0.1 mg/kg to about 30 mg/kg, from about 1 mg/kg to about 30mg/kg, from about 3 mg/kg to about 30 mg/kg, from about 3 mg/kg to about25 mg/kg, from about 3 mg/kg to about 20 mg/kg, from about 5 mg/kg toabout 15 mg/kg, or from about 7 mg/kg to about 12 mg/kg. It isrecognized that the treatment may comprise administration of a singletherapeutically effective dose or administration of multipletherapeutically effective doses of the anti-CD40 antibody orantigen-binding fragment thereof.

[0013] The anti-CD40 antibodies suitable for use in the methods of theinvention may be modified Modifications of the anti-CD40 antibodiesinclude, but are not limited to, immunologically active chimericanti-CD40 antibodies, humanized anti-CD40 antibodies, andimmunologically active murine anti-CD40 antibodies.

BRIEF DESCRIPTION OF THE DRAWINGS

[0014]FIG. 1 depicts representative results of the effect of agonist(MS81) and antagonist (15B8) anti-CD40 antibodies at a concentration of1, 2, or 5 μg/ml on the proliferation of non-Hodgkin's lymphoma (NHL)cells in vitro in the absence of interleukin-4 (IL-4). Malignant B cellswere obtained from tumor infiltrated lymph nodes of a NHL patient. FACSanalysis of the NHL cells confirmed that these cells expressed CD40 andbound the antagonist anti-CD40 antibody 15B8. See Example 3 below fordetails.

[0015]FIG. 2 depicts representative results of the effect of agonist(MS81) and antagonist (15B8) anti-CD40 antibodies at a concentration of1, 2, or 5 μg/ml on the proliferation of non-Hodgkin's lymphoma (NHL)cells in vitro in the presence of IL-4(2 ng/ml). Malignant B cells wereobtained from tumor infiltrated lymph nodes of a NHL patient. FACSanalysis of the NHL cells confirmed that these cells expressed CD40 andbound the antagonist anti-CD40 antibody. See Example 3 below fordetails.

[0016]FIG. 3 depicts representative results of the effect of agonist(MS81) and antagonist (15B8) anti-CD40 antibodies at a concentration of1, 2, or 5 μg/ml on CD40L-stimulated proliferation of NHL cells in vitroin the absence of IL-4. The NHL cells were obtained from aRituximab-sensitive NHL patient. See Example 4 below for details.

[0017]FIG. 4 depicts a representative dose response curve for theantagonist anti-CD40 antibody 15B8 on proliferation of NHL cellsstimulated in vitro by CD40L and IL-4 (2 ng/ml). The NHL cells wereobtained from a Rituximab-sensitive NHL patient. See Example 4 below fordetails.

[0018]FIG. 5 depicts dose response curves for the antagonist anti-CD40antibody 15B8 on proliferation of purified human peripheral blood Bcells stimulated in vitro in a CD40L-expressing CHO cell-mediated humanB-cell proliferation assay. The B cells were obtained from 3 healthyindividuals. See Example 6 below for details.

[0019]FIG. 6 depicts the effect on the peripheral B-cell count in malechimpanzees after administration of 15B8 at doses of 0.03 or 3 mg/kg.Each dosage level was intravenously administered to 3 chimpanzees, andthe average peripheral B-cell count (per μl) was determined (righty-axis). The mean concentration of 15B8 in the serum (ng/ml) is depictedon the left y-axis. Time measured in days relative to the IVadministration is shown on the x-axis. After administration of 15B8 at 3mg/kg, serum 15B8 concentrations declined in a triphasic pattern with ashort distribution phase, a log-linear elimination phase, and anon-linear elimination phase. The half-life during the log-linearelimination phase was approximately 4 days. Peripheral B-cell numbersdecreased immediately after 15B8 administration and recovered within 3-4weeks. See Example 9 below for details.

DETAILED DESCRIPTION OF THE INVENTION

[0020] The present invention is directed to methods for treating humanpatients with diseases that originate from malignant B cells. Themethods involve treatment with an anti-CD40 antibody or antigen-bindingfragment thereof, where administration of the antibody orantigen-binding fragment thereof promotes a positive therapeuticresponse within the patient undergoing this method of therapy. Anti-CD40antibodies suitable for use in the methods of the invention have thefollowing characteristics: 1) they specifically bind a human CD40antigen expressed on the surface of a human cell; 2) they are free ofsignificant agonist activity when bound to a CD40 antigen on a normalhuman B cell; and 3) they exhibit antagonist activity when bound to aCD40 antigen on a malignant human B cell. These anti-CD40 antibodies andantigen-binding fragments thereof are referred to herein as antagonistanti-CD40 antibodies. Such antibodies include, but are not limited to,the fully human monoclonal antibody 15B8 described below and monoclonalantibodies having the binding characteristics of monoclonal antibody15B8. As discussed in more detail below, these antibodies are specificto CD40 receptors. When these antibodies bind CD40 displayed on thesurface of normal human B cells, the antibodies are free of significantagonist activity, in some embodiments, their binding to CD40 displayedon the surface of normal human B cells results in inhibition ofproliferation and differentiation of these normal human B cells. Thus,the antagonist anti-CD40 antibodies suitable for use in the methods ofthe invention include those monoclonal antibodies that can exhibitantagonist activity toward normal human B cells expressing thecell-surface CD40 antigen. When antagonist anti-CD40 antibodies bindCD40 displayed on the surface of malignant human B cells, the antibodiesexhibit antagonist activity as defined elsewhere herein.

[0021] “Treatment” is herein defined as the application oradministration of an antagonist anti-CD40 antibody or antigen-bindingfragment thereof to a patient, or application or administration of anantagonist anti-CD40 antibody or fragment thereof to an isolated tissueor cell line from a patient, where the patient has a disease, a symptomof a disease, or a predisposition toward a disease, where the purpose isto cure, heal, alleviate, relieve, alter, remedy, ameliorate, improve,or affect the disease, the symptoms of the disease, or thepredisposition toward the disease. By “treatment” is also intended theapplication or administration of a pharmaceutical composition comprisingthe antagonist anti-CD40 antibodies or fragments thereof to a patient,or application or administration of a pharmaceutical compositioncomprising the anti-CD40 antibodies or fragments thereof to an isolatedtissue or cell line from a patient, who has a disease, a symptom of adisease, or a predisposition toward a disease, where the purpose is tocure, heal, alleviate, relieve, alter, remedy, ameliorate, improve, oraffect the disease, the symptoms of the disease, or the predispositiontoward the disease.

[0022] By “anti-tumor activity” is intended a reduction in the rate ofmalignant B-cell proliferation or accumulation, and hence a decline ingrowth rate of an existing tumor or in a tumor that arises duringtherapy, and/or destruction of existing neoplastic (tumor) cells ornewly formed neoplastic cells, and hence a decrease in the overall sizeof a tumor during therapy. Therapy with at least one anti-CD40 antibody(or antigen-binding fragment thereof) causes a physiological responsethat is beneficial with respect to treatment of disease statescomprising malignant B cells in a human.

[0023] The monoclonal antibody 15B8 represents a suitable antagonistanti-CD40 antibody for use in the methods of the present invention. Thisantibody is described in U.S. Provisional Application Serial No.60/237,556, titled “Human Anti-CD40 Antibodies,” filed Oct. 2, 2000, andPCT International Application No. PCT/US01/______, also titled “HumanAnti-CD40 Antibodies,” filed Oct. 2, 2001 (Attorney Docket No.PP16092.003), both of which are herein incorporated by reference intheir entirety. The 15B8 antibody is a fully human anti-CD40 monoclonalantibody of the IgG₂ isotype produced from the hybridoma cell line 15B8.The cell line was created using splenocytes from an immunized xenotypicmouse containing a human immunoglobulin locus (Abgenix). The spleencells were fused with the mouse myeloma SP2/0 cells (Sierra BioSource).The resulting hybridomas were sub-cloned several times to create thestable monoclonal cell line 15B8.

[0024] The 15B8 cell line was adapted to grow in protein-free medium andused to create a Master Cell Bank. The Master Cell Bank was tested foridentity and adventitious and endogenous contaminants. The Master CellBank was used to manufacture the desired human IgG₂. The respective 15B8antibody was purified using chromatography and filtration procedures.

[0025] The anti-CD40 antibody 15B8 is a polypeptide composed of 1,284amino acid residues with a predicted molecular weight of 149,755 withtwo heavy chains and two light chains in a heterodineric arrangement.Amino acid analysis reveals that the antibody is composed of equimolaramounts of heavy and light chains. The nucleotide and amino acidsequences for the variable region for the light chain are set forth inSEQ ID NO:1 and SEQ ID NO:2, respectively. The nucleotide and amino acidsequences for the variable region for the heavy chain are set forth inSEQ ID NO:3 and SEQ ID NO:4, respectively. The 15B8 monoclonal antibodybinds soluble CD40 in ELISA-type assays. When tested in vitro foreffects on proliferation of B cells from numerous primates, 15B8 acts asan agonistic anti-CD40 antibody in cynomologus, baboon, and rhesusmonkeys. In assays with humans, chimpanzees, and marmosets, 15B8 is anantagonist anti-CD40 antibody. The binding affinity of 15B8 to humanCD40 is 3.1×10⁻⁹M as determined by the Biacore™ assay.

[0026] Suitable antagonist anti-CD40 antibodies for use in the methodsof the present invention exhibit a strong single-site binding affinityfor the CD40 cell-surface antigen. The monoclonal antibodies of theinvention exhibit a dissociation constant (K_(d)) for CD40 of at least10⁻⁵ M, at least 3×10⁻⁵ M, preferably at least 10⁻⁶ M to 10⁻⁷ M, morepreferably at least 10⁻⁸ M to about 10⁻²⁰ M, yet more preferably atleast 5×10⁻⁹ M to about 10⁻¹⁸ M, most preferably at least about 5×10⁻⁹ Mto about 10⁻¹⁶ M, such as 10⁻⁸ M, 5×10⁻⁹ M, 10⁻⁹ M, 5×10⁻¹⁰ M, 10⁻¹⁰ M,5×10⁻¹¹ M, 10⁻¹¹ M, 5×10⁻¹² M, 10⁻¹² M, 5×10⁻¹³ M, 10⁻¹³ M, 5×10⁻¹⁴ M,10⁻¹⁴ M, 5×10⁻¹⁵ M, 10⁻¹⁵ M, 5×10⁻¹⁶ M, or 10⁻¹⁶ M, as measured using astandard assay such as Biacore™ Biacore analysis is known in the art anddetails are provided in the “BIAapplications handbook.”

[0027] By “CD40 antigen” is intended a glycosylated transmembranepeptide or any fragment thereof (GenBank Accession No. X60592; U.S. Pat.Nos. 5,674,492 and 4,708,871; Stamenkovic et al. (1989) EMBO 8:1403;Clark (1990) Tissue Antigens 36:33; Barclay et al. (1997) The LeucocyteAntigen Facts Book (2d ed.; Academic Press, San Diego)). The CD40receptor is displayed on the surface of a variety of cell types, asdescribed elsewhere herein. By “displayed on the surface” and “expressedon the surface” is intended that all or a portion of the CD40 antigen isexposed to the exterior of the cell. The displayed or expressed CD40antigen may be fully or partially glycosylated.

[0028] By “agonist activity” is intended that the substance functions asan agonist. An agonist combines with a receptor on a cell and initiatesa reaction or activity that is similar to or the same as that initiatedby the receptor's natural ligand. An agonist of CD40 induces any or allof, but not limited to, the following responses: B-cell proliferationand differentiation, antibody production, intercellular adhesion, B-cellmemory generation, isotype switching, up-regulation of cell-surfaceexpression of MHC Class II and CD80/86, and secretion ofpro-inflammatory cytokines such as IL-8, IL-12, and TNF. By “antagonistactivity” is intended that the substance functions as an antagonist. Anantagonist of CD40 prevents or reduces induction of any of the responsesinduced by binding of the CD40 receptor to an agonist ligand,particularly CD40L. The antagonist may reduce induction of any one ormore of the responses to agonist binding by 5%, 10%, 15%, 20%, 25%, 30%,35%, preferably 40%, 45%, 50%, 55%, 60%, more preferably 70%, 80%, 85%,and most preferably 90%, 95%, 99%, or 100%. Methods for measuring B-cellresponses are known to one of skill in the art and include, but are notlimited to, B-cell proliferation assays, Banchereau-Like-B-Cellproliferation assays, T-cell helper assays for antibody production,co-stimulation of B-cell proliferation assays, and assays forup-regulation of B-cell activation markers. Several of these assays arediscussed in more detail elsewhere herein.

[0029] By “significant” agonist activity is intended an agonist activityof at least 30%, 35%, 40%, 45%, 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%,or 100% greater than the agonist activity induced by a neutral substanceor negative control as measured in an assay of a B-cell response. Asubstance “free of significant agonist activity” would exhibit anagonist activity of not more than about 25% greater than the agonistactivity induced by a neutral substance or negative control, preferablynot more than about 20% greater, 15% greater, 10% greater, 5% greater,1% greater, 0.5% greater, or even not more than about 0.1% greater thanthe agonist activity induced by a neutral substance or negative controlas measured in an assay of a B-cell response. The antagonist anti-CD40antibodies useful in the methods of the present invention are free ofsignificant agonist activity as noted above when bound to a CD40 antigenon a normal human B cell. In one embodiment of the invention, theantagonist anti-CD40 antibody is free of significant agonist activity inone B-cell response. In another embodiment of the invention, theantagonist anti-CD40 antibody is free of significant agonist activity inassays of more than one B-cell response (e.g., proliferation anddifferentiation, or proliferation, differentiation, and antibodyproduction).

[0030] As used herein “anti-CD40 antibody” encompasses any antibody thatspecifically recognizes the CD40 B-cell surface antigen, includingpolyclonal antibodies, monoclonal antibodies, single-chain antibodies,and fragments thereof such as Fab, F(ab′)₂, F_(v), and other fragmentswhich retain the antigen binding function of the parent anti-CD40antibody. Polyclonal sera may be prepared by conventional methods. Ingeneral, a solution containing the CD40 antigen is first used toimmunize a suitable animal, preferably a mouse, rat, rabbit, or goat.Rabbits or goats are preferred for the preparation of polyclonal seradue to the volume of serum obtainable, and the availability of labeledanti-rabbit and anti-goat antibodies. Polyclonal sera can be prepared ina transgenic animal, preferably a mouse bearing human immunoglobulinloci. In a preferred embodiment, Sf9 cells expressing CD40 are used asthe immunogen. Immunization can also be performed by mixing oremulsifying the antigen-containing solution in saline, preferably in anadjuvant such as Freund's complete adjuvant, and injecting the mixtureor emulsion parenterally (generally subcutaneously or intramuscularly).A dose of 50-200 μg/injection is typically sufficient. Immunization isgenerally boosted 2-6 weeks later with one or more injections of theprotein in saline, preferably using Freund's incomplete adjuvant. Onemay alternatively generate antibodies by in vitro immunization usingmethods known in the art, which for the purposes of this invention isconsidered equivalent to in vivo immunization. Polyclonal antisera areobtained by bleeding the immunized animal into a glass or plasticcontainer, incubating the blood at 25° C. for one hour, followed byincubating at 4° C. for 2-18 hours. The serum is recovered bycentrifugation (e.g., 1,000×g for 10 minutes). About 20-50 ml per bleedmay be obtained from rabbits.

[0031] Preferably the antibody is monoclonal in nature. By “monoclonalantibody” is intended an antibody obtained from a population ofsubstantially homogeneous antibodies, i.e., the individual antibodiescomprising the population are identical except for possible naturallyoccurring mutations that may be present in minor amounts. Monoclonalantibodies are highly specific, being directed against a singleantigenic site, i.e., the CD40 B-cell surface antigen in the presentinvention. Furthermore, in contrast to conventional (polyclonal)antibody preparations that typically include different antibodiesdirected against different determinants (epitopes), each monoclonalantibody is directed against a single determinant on the antigen. Themodifier “monoclonal” indicates the character of the antibody as beingobtained from a substantially homogeneous population of antibodies, andis not to be construed as requiring production of the antibody by anyparticular method. For example, the monoclonal antibodies to be used inaccordance with the present invention may be made by the hybridomamethod first described by Kohler et al. (1975) Nature 256:495, or may bemade by recombinant DNA methods (see, e.g., U.S. Pat. No. 4,816,567).The “monoclonal antibodies” may also be isolated from phage antibodylibraries using the techniques described in, for example, Clackson etal. (1991) Nature 352:624-628; Marks et al. (1991) J. Mol. Biol.222:581-597; and U.S. Pat. No. 5,514,548.

[0032] Monoclonal antibodies can be prepared using the method of Kohleret al. (1975) Nature 256:495-496, or a modification thereof. Typically,a mouse is immunized with a solution containing an antigen. Immunizationcan be performed by mixing or emulsifying the antigen-containingsolution in saline, preferably in an adjuvant such as Freund's completeadjuvant, and injecting the mixture or emulsion parenterally. Any methodof immunization known in the art may be used to obtain the monoclonalantibodies of the invention. After immunization of the animal, thespleen (and optionally, several large lymph nodes) are removed anddissociated into single cells. The spleen cells may be screened byapplying a cell suspension to a plate or well coated with the antigen ofinterest. The B cells expressing membrane bound immunoglobulin specificfor the antigen bind to the plate and are not rinsed away. Resulting Bcells, or all dissociated spleen cells, are then induced to fuse withmyeloma cells to form hybridomas, and are cultured in a selectivemedium. The resulting cells are plated by serial dilution and areassayed for the production of antibodies that specifically bind theantigen of interest (and that do not bind to unrelated antigens). Theselected monoclonal antibody (mAb)-secreting hybridomas are thencultured either in vitro (e.g., in tissue culture bottles or hollowfiber reactors), or in vivo (as ascites in mice).

[0033] As an alternative to the use of hybridomas, antibody can beproduced in a cell line such as a CHO cell line, as disclosed in U.S.Pat. Nos. 5,545,403; 5,545,405; and 5,998,144; incorporated herein byreference. Briefly the cell line is transfected with vectors capable ofexpressing a light chain and a heavy chain, respectively. Bytransfecting the two proteins on separate vectors, chimeric antibodiescan be produced. Another advantage is the correct glycosylation of theantibody.

[0034] Monoclonal antibodies to CD40 are known in the art. See, forexample, the sections dedicated to B-cell antigen in McMichael, ed.(1987; 1989) Leukocyte Typing III and IV (Oxford University Press, NewYork); U.S. Pat. Nos. 5,674,492; 5,874,082; 5,677,165; 6,056,959; WO00/63395; copending U.S. Provisional Patent Application Serial No.60/237,556, titled, “Human Anti-CD40 Antibodies,” filed Oct. 2, 2000;Gordon et al. (1988) J. Immunol. 140:1425; Valle et al (1989) Eur. J.Immunol. 19:1463; Clark et al. (1986) PNAS 83:4494; Paulie et al (1989)J. Immunol. 142:590; Gordon et al. (1987) Eur. J. Immunol. 17:1535;Jabara et al. (1990) J. Exp. Med. 172:1861; Zhang et al (1991) J.Immunol. 146:1836; Gascan et al. (1991) J. Immunol. 147:8; Banchereau etal (1991) Clin. Immunol. Spectrum 3:8; and Banchereau et al. (1991)Science 251:70; all of which are herein incorporated by reference.

[0035] Additionally, the term “anti-CD40 antibody” as used hereinencompasses chimeric anti-CD40 antibodies. By “chimeric” antibodies isintended antibodies that are most preferably derived using recombinantdeoxyribonucleic acid techniques and which comprise both human(including immunologically “related” species, e.g., chimpanzee) andnon-human components. Thus, the constant region of the chimeric antibodyis most preferably substantially identical to the constant region of anatural human antibody, the variable region of the chimeric antibody ismost preferably derived from a non-human source and has the desiredantigenic specificity to the CD40 cell-surface antigen. The non-humansource can be any vertebrate source that can be used to generateantibodies to a human CD40 cell-surface antigen or material comprising ahuman CD40 cell-surface antigen. Such non-human sources include, but arenot limited to, rodents (e.g., rabbit, rat, mouse, etc.; see, forexample, U.S. Pat. No. 4,816,567, herein incorporated by reference) andnon-human primates (e.g., Old World Monkey, Ape, etc.; see, for example,U.S. Pat. Nos. 5,750,105 and 5,756,096; herein incorporated byreference). As used herein, the phrase “immunologically active” whenused in reference to chimeric anti-CD40 antibodies means a chimericantibody that binds human CD40.

[0036] Humanized anti-CD40 antibodies are also encompassed by the termanti-CD40 antibody as used herein. By “humanized” is intended forms ofanti-CD40 antibodies that contain minimal sequence derived fromnon-human immunoglobulin sequences. For the most part, humanizedantibodies are human immunoglobulins (recipient antibody) in whichresidues from a hypervariable region (also known as complementaritydetermining region or CDR) of the recipient are replaced by residuesfrom a hypervariable region of a non-human species (donor antibody) suchas mouse, rat, rabbit, or nonhuman primate having the desiredspecificity, affinity, and capacity. Humanization can be essentiallyperformed following the method of Winter and co-workers (Jones et al.(1986) Nature 321:522-525; Riechmann et al. (1988) Nature 332:323-327;Verhoeyen et al. (1988) Science 239:15341536), by substituting rodent ormutant rodent CDRs or CDR sequences for the corresponding sequences of ahuman antibody. See also U.S. Pat. Nos. 5,225,539; 5,585,089; 5,693,761;5,693,762; 5,859,205; herein incorporated by reference. In someinstances, residues within the framework regions of one or more variableregions of the human immunoglobulin are replaced by correspondingnon-human residues (see, for example, U.S. Pat. Nos. 5,585,089;5,693,761; 5,693,762; and 6,180,370). Furthermore, humanized antibodiesmay comprise residues that are not found in the recipient antibody or inthe donor antibody. These modifications are made to further refineantibody performance (e.g., to obtain desired affinity). In general, thehumanized antibody will comprise substantially all of at least one, andtypically two, variable domains, in which all or substantially all ofthe hypervariable regions correspond to those of a non-humanimmunoglobulin and all or substantially all of the framework regions arethose of a human immunoglobulin sequence. The humanized antibodyoptionally also will comprise at least a portion of an immunoglobulinconstant region (Fc), typically that of a human immunoglobulin. Forfurther details see Jones et al. (1986) Nature 331:522-525; Riechmann etal. (1988) Nature 332:323-329; and Presta (1992) Curr. Op. Struct. Biol.2:593-596; herein incorporated by reference. Accordingly, such“humanized” antibodies may include antibodies wherein substantially lessthan an intact human variable domain has been substituted by thecorresponding sequence from a non-human species. In practice, humanizedantibodies are typically human antibodies in which some CDR residues andpossibly some framework residues are substituted by residues fromanalogous sites in rodent antibodies. See, for example, U.S. Pat. Nos.5,225,539; 5,585,089; 5,693,761; 5,693,762; 5,859,205. See also U.S.Pat. No. 6,180,370, and International Publication No. WO 01/27160, wherehumanized antibodies and techniques for producing humanized antibodieshaving improved affinity for a predetermined antigen are disclosed.

[0037] Also encompassed by the term anti-CD40 antibodies are xenogeneicor modified anti-CD40 antibodies produced in a non-human mammalian host,more particularly a transgenic mouse, characterized by inactivatedendogenous immunoglobulin (Ig) loci. In such transgenic animals,competent endogenous genes for the expression of light and heavysubunits of host immunoglobulins are rendered non-functional andsubstituted with the analogous human immunoglobulin loci. Thesetransgenic animals produce human antibodies in the substantial absenceof light or heavy host innmunoglobulin subunits. See, for example, U.S.Pat. Nos. 5,877,397 and 5,939,598, herein incorporated by reference.

[0038] Fragments of the anti-CD40 antibodies are suitable for use in themethods of the invention so long as they retain the desired affinity ofthe full-length antibody. Thus, a fragment of an anti-CD40 antibody willretain the ability to bind to the CD40 B-cell surface antigen. Suchfragments are characterized by properties similar to the correspondingfull-length antagonist anti-CD40 antibody, that is the fragments will 1)specifically bind a human CD40 antigen expressed on the surface of ahuman cell; 2) are free of significant agonist activity when bound to aCD40 antigen on a normal human B cell; and 3) exhibit antagonistactivity when bound to a CD40 antigen on a malignant human B cell. Wherethe full-length antagonist anti-CD40 antibody exhibits antagonistactivity when bound to the CD40 antigen on the surface of a normal humanB cell, the fragment will also exhibit such antagonist activity. Suchfragments are referred to herein as “antigen-binding” fragments.

[0039] Suitable antigen-binding fragments of an antibody comprise aportion of a full-length antibody, generally the antigen-binding orvariable region thereof. Examples of antibody fragments include, but arenot limited to, Fab, F(ab′)₂, and Fv fragments and single-chain antibodymolecules. By “single-chain Fv” or “sFv” antibody fragments is intendedfragments comprising the V_(H) and V_(L) domains of an antibody, whereinthese domains are present in a single polypeptide chain. See, forexample, U.S. Pat. Nos. 4,946,778; 5,260,203; 5,455,030; 5,856,456;herein incorporated by reference. Generally, the Fv polypeptide furthercomprises a polypeptide linker between the V_(H) and V_(L) domains thatenables the sFv to form the desired structure for antigen binding. For areview of sFv see Pluckthun (1994) in The Pharmacology of MonoclonalAntibodies, Vol. 113, ed. Rosenburg and Moore (Springer-Verlag, NewYork), pp. 269-315.

[0040] Antibodies or antibody fragments can be isolated from antibodyphage libraries generated using the techniques described in, forexample, McCafferty et al. (1990) Nature 348:552-554 (1990) and U.S.Pat. No. 5,514,548. Clackson et al. (1991) Nature 352:624-628 and Markset al. (1991) J. Mol. Biol. 222:581-597 describe the isolation of murineand human antibodies, respectively, using phage libraries. Subsequentpublications describe the production of high affinity (nM range) humanantibodies by chain shuffling (Marks et al. (1992) Bio/Technology10:779-783), as well as combinatorial infection and in vivorecombination as a strategy for constructing very large phage libraries(Waterhouse et al. (1993) Nucleic. Acids Res. 21:2265-2266). Thus, thesetechniques are viable alternatives to traditional monoclonal antibodyhybridoma techniques for isolation of monoclonal antibodies.

[0041] Various techniques have been developed for the production ofantibody fragments. Traditionally, these fragments were derived viaproteolytic digestion of intact antibodies (see, e.g., Morimoto et al.(1992) Journal of Biochemical and Biophysical Methods 24:107-117 (1992)and Brenman et al. (1985) Science 229:81). However, these fragments cannow be produced directly by recombinant host cells. For example, theantibody fragments can be isolated from the antibody phage librariesdiscussed above. Alternatively, Fab′-SH fragments can be directlyrecovered from E. coli and chemically coupled to form F(ab′)₂ fragments(Carter et al. (1992) Bio/Technology 10:163-167). According to anotherapproach, F(ab′)₂ fragments can be isolated directly from recombinanthost cell culture. Other techniques for the production of antibodyfragments will be apparent to the skilled practitioner.

[0042] Antagonist anti-CD40 antibodies useful in the methods of thepresent invention include the 15B8 monoclonal antibody disclosed hereinas well as antibodies differing from this antibody but retaining theCDRS; and antibodies with one or more amino acid addition(s),deletion(s), or substitution(s), wherein the antagonist activity ismeasured by inhibition of malignant B cell proliferation and/ordifferentiation. The invention also encompasses de-immunized antagonistanti-CD40 antibodies, which can be produced as described in, forexample, International Publication Nos. WO 98/52976 and WO 0034317;herein incorporated by reference. In this manner, residues within theantagonist anti-CD40 antibodies of the invention are modified so as torender the antibodies non- or less immunogenic to humans while retainingtheir antagonist activity toward malignant human B cells, wherein suchactivity is measured by assays noted elsewhere herein. Also includedwithin the scope of the claims are fusion proteins comprising anantagonist anti-CD40 antibody of the invention, or a fragment thereof,which fusion proteins can be synthesized or expressed from correspondingpolynucleotide vectors, as is known in the art. Such fusion proteins aredescribed with reference to conjugation of antibodies as noted below.

[0043] The antibodies of the present invention can have sequencevariations produced using methods described in, for example, PatentPublication Nos. EP 0 983 303 A1, WO 00/34317, and WO 98/52976,incorporated herein by reference. For example, it has been shown thatsequences within the CDR can cause an antibody to bind to MHC Class IIand trigger an unwanted helper T cell response. A conservativesubstitution can allow the antibody to retain binding activity yet loseits ability to trigger an unwanted T cell response. Any suchconservative or non-conservative substitutions can be made usingart-recognized methods, such as those noted elsewhere herein, and theresulting antibodies will fall within the scope of the invention. Thevariant antibodies can be routinely tested for antagonist activity,affinity, and specificity using methods described herein.

[0044] An antibody produced by any of the methods described above, orany other method not disclosed herein, will fall within the scope of theinvention if it possesses at least one of the following biologicalactivities: inhibition of immunoglobulin secretion by normal humanperipheral B cells stimulated by T cells; inhibition of proliferation ofnormal human peripheral B cells stimulated by Jurkat T cells; inhibitionof proliferation of normal human peripheral B cells stimulated byCD40L-expressing cells; and inhibition of proliferation of humanmalignant B cells as noted below. These assays can be performed asdescribed in the Examples herein. See also the assays described inSchultze et al. (1998) Proc. Natl. Acad. Sci. USA 92:8200-8204; Dentonet al. (1998) Pediair Transplant. 2:6-15; Evans et al. (2000) J.Immunol. 164:688-697; Noelle (1998) Agents Actions Suppl. 49:17-22;Lederman et al. (1996) Curr. Opin. Hematol. 3:77-86; Coligan et al.(1991) Current Protocols in Immunology 13:12; Kwekkeboom et al. (1993)Immunology 79:439-444; and U.S. Pat. Nos. 5,674,492 and 5,847,082;herein incorporated by reference.

[0045] Any of the previously described antagonist anti-CD40 antibodiesor antibody fragments thereof may be conjugated prior to use in themethods of the present invention Methods for producing conjugatedantibodies are known in the art. Thus, the anti-CD40 antibody may belabeled using an indirect labeling or indirect labeling approach By“indirect labeling” or “indirect labeling approach” is intended that achelating agent is covalently attached to an antibody and at least oneradionuclide is inserted into the chelating agent. See, for example, thechelating agents and radionuclides described in Srivagtava and Mease(1991) Nucl. Med. Bio. 18:589-603, herein incorporated by reference.Alternatively, the anti-CD40 antibody may be labeled using “directlabeling” or a “direct labeling approach”, where a radionuclide iscovalently attached directly to an antibody (typically via an amino acidresidue). Preferred radionuclides are provided in Srivagtava and Mease(1991) supra. The indirect labeling approach is particularly preferred.See also, for example, International Publication Nos. WO 00/52031 and WO00/52473, where a linker is used to attach a radioactive label toantibodies; and the labeled forms of anti-CD40 antibodies described inU.S. Pat. No. 6,015,542; herein incorporated by reference.

[0046] Further, an antibody (or fragment thereof) may be conjugated to atherapeutic moiety such as a cytotoxin, a therapeutic agent, or aradioactive metal ion. A cytotoxin or cytotoxic agent includes any agentthat is detrimental to cells. Examples include taxol, cytochalasin B,gramicidin D, ethidium bromide, emetine, mitomycin, etoposide,tenoposide, vincristine, vinblastine, colchicin, doxorubicin,daunombicin, dihydroxy anthracin dione, mitoxantrone, mithramycin,actinomycin D, 1-dehydrotestosterone, glucocorticoids, procaine,tetracaine, lidocaine, propranolol, and puromycin and analogs orhomologs thereof. Therapeutic agents include, but are not limited to,antimetabolites (e.g., methotrexate, 6-mercaptopurine, 6-thioguanine,cytarabine, 5-fluorouracil decarbazine), alkylating agents (e.g.,mechlorethamine, thioepa chlorambucil, melphalan, carmustine (BSNU) andlomustine (CCNU), cyclothosphamide, busulfan, dibromomannitol,streptozotocin, mitomycin C, and cis-dichlorodiamine platinum (II) (DDP)cisplatin), anthracyclines (e.g., daunorubicin (formerly daunomycin) anddoxorubicin), antibiotics (e.g., dactinomycin (formerly actinomycin),bleomycin, mithramycin, and anthramycin (AMC)), and anti-mitotic agents(e.g., vincristine and vinblastine). The conjugates of the invention canbe used for modifying a given biological response; the drug moiety isnot to be construed as limited to classical chemical therapeutic agents.For example, the drug moiety may be a protein or polypeptide possessinga desired biological activity. Such proteins may include, for example, atoxin such as abrin, ricin A, pseudomonas exotoxin, or diphtheria toxin;a protein such as tumor necrosis factor, interferon-alpha,interferon-beta, nerve growth factor, platelet derived growth factor,tissue plasminogen activator; or, biological response modifiers such as,for example, lymphokines, interleukin-1 (“IL-1”), interleukin-2(“IL-2”), interleukin-6 (“IL-6”), granulocyte macrophase colonystimulating factor (“GM-CSF”), granulocyte colony stimulating factor(“G-CSF”), or other growth factors.

[0047] Techniques for conjugating such therapeutic moiety to antibodiesare well known. See, for example, Arnon et al. (1985) “MonoclonalAntibodies for Immunotargeting of Drugs in Cancer Therapy,” inMonoclonal Antibodies and Cancer Therapy, ed. Reisfeld et al. (Alan R.Liss, Inc.), pp. 243-25⁶; ed. Hellstrom et al. (1987) “Antibodies forDrug Delivery,” in Controlled Drug Delivery, ed. Robinson et al. (2d ed;Marcel Dekker, Inc.), pp. 623-653; Thorpe (1985) “Antibody Carriers ofCytotoxic Agents in Cancer Therapy: A Review,” in Monoclonal Antibodies'84: Biological and Clinical Applications, ed. Pinchera et al. pp.475-506 (Editrice Kurtis, Milano, Italy, 1985); “Analysis, Results, andFuture Prospective of the Therapeutic Use of Radiolabeled Antibody inCancer Therapy,” in Monoclonal Antibodiesfor Cancer Detection andTherapy, ed. Baldwin et al. (Academic Press, New York, 1985), pp.303-316; and Thorpe et al. (1982) Immunol. Rev. 62:119-158.

[0048] Alternatively, an antibody can be conjugated to a second antibodyto form an antibody heteroconjugate as described by Segal in U.S. Pat.No. 4,676,980. In addition, linkers may be used between the labels andthe antibodies of the invention (see U.S. Pat. No. 4,831,175).Antibodies or, antigen-binding fragments thereof may be directly labeledwith radioactive iodine, indium, yttrium, or other radioactive particleknown in the art (U.S. Pat. No. 5,595,721). Treatment may consist of acombination of treatment with conjugated and nonconjugated antibodiesadministered simultaneously or subsequently (WO 00/52031 and WO00/52473).

[0049] Methods of the invention are directed to the use of antagonistanti-CD40 antibodies to treat patients having a disease comprisingmalignant B cells. By “malignant” B cell is intended any neoplastic Bcell, including but not limited to B cells derived from lymphomasincluding low-, intermediate-, and high-grade B-cell lymphomas,immunoblastic lymphomas, non-Hodgkin's lymphomas, Hodgkin's disease,Epstein-Barr Virus (EBV) induced lymphomas, and AIDS-related lymphomas,as well as B-cell acute lymphoblastic leukemias, myelomas, chroniclymphocytic leukemias, acute myeloblastic leukemias, and the like.

[0050] The methods of the invention find use in the treatment ofnon-Hodgkin's lymphomas related to abnormal, uncontrollable B cellproliferation or accumulation. For purposes of the present invention,such lymphomas will be referred to according to the Working Formulationclassification scheme, that is those B-cell lymphomas categorized as lowgrade, intermediate grade, and high grade (see “The Non-Hodgkin'sLymphoma Pathologic Classification Project,” Cancer 49(1982):2112-2135).Thus, low-grade B-cell lymphomas include small lymphocytic, follicularsmall-cleaved cell, and follicular mixed small-cleaved and large celllymphomas; intermediate-grade lymphomas include follicular large cell,diffuse small cleaved cell, diffuse mixed small and large cell, anddiffuse large cell lymphomas; and high-grade lymphomas include largecell immunoblastic, lymphoblastic, and small non-cleaved cell lymphomasof the Burkitt's and non-Burkitt's type.

[0051] It is recognized that the methods of the invention are useful inthe therapeutic treatment of B-cell lymphomas that are classifiedaccording to the Revised European and American Lymphoma Classification(REAL) system. Such B-cell lymphomas include, but are not limited to,lymphomas classified as precursor B-cell neoplasms, such asB-lymphoblastic leukemia/lymphoma; peripheral B-cell neoplasms,including B-cell chronic lymphocytic leukemia/small lymphocyticlymphoma, lymphoplasmacytoid lymphoma/immunocytoma, mantle cell lymphoma(MCL), follicle center lymphoma (follicular) (including diffuse smallcell, diffuse mixed small and large cell, and diffuse large celllymphomas), marginal zone B-cell lymphoma (including extranodal, nodal,and splenic types), hairy cell leukemia, plasmacytoma/myeloma, diffuselarge cell B-cell lymphoma of the subtype primary mediastinal (thymic),Burkitt's lymphoma, and Burkitt's like high grade B-cell lymphoma; acuteleukemias; acute lymphocytic leukemias; myeloblastic leukemias; acutemyelocytic leukemias; promyelocytic leukemia; myelomonocytic leukemia;monocytic leukemia; erythroleukemia; granulocytic leukemia (chronicmyelocytic leukemia); chronic lymphocytic leukemia; polycythemia vera;multiple myeloma; Waldenstrom's macroglobulinemia; heavy chain disease;and unclassifiable low-grade or high-grade B-cell lymphomas.

[0052] It is recognized that the methods of the invention may be usefulin preventing further tumor outgrowths arising during therapy. Themethods of the invention are particularly useful in the treatment ofsubjects having low-grade B-cell lymphomas, particularly those subjectshaving relapses following standard chemotherapy. Low-grade B-celllymphomas are more indolent than the intermediate- and high-grade B-celllymphomas and are characterized by a relapsing/remitting course. Thus,treatment of these lymphomas is improved using the methods of theinvention, as relapse episodes are reduced in number and severity.

[0053] The antagonist anti-CD40 antibodies described herein may alsofind use in the treatment of inflammatory diseases and deficiencies ordisorders of the immune system including, but not limited to, systemiclupus erythematosus,psoriasis, scleroderma, CREST syndrome, inflammatorymyositis, Sjogren's syndrome, mixed connective tissue disease,rheumatoid arthritis, multiple sclerosis, inflammatory bowel disease,acute respiratory distress syndrome, pulmonary inflammation, idiopathicpulmonary fibrosis, osteoporosis, delayed type hypersensitivity, asthma,primary biliary cirrhosis, and idiopathic thrombocytopenic purpura.

[0054] In accordance with the methods of the present invention, at leastone antagonist anti-CD40 antibody (or antigen-binding fragment thereof)as defined elsewhere herein is used to promote a positive therapeuticresponse with respect to a malignant human B cell. By “positivetherapeutic response” is intended an improvement in the disease inassociation with the anti-tumor activity of these antibodies orfragments thereof, and/or an improvement in the symptoms associated withthe disease. That is, an anti-proliferative effect, the prevention offurther tumor outgrowths, and/or a decrease in B symptoms can beobserved. Thus, for example, an improvement in the disease may becharacterized as a complete response. By “complete response” is intendedan absence of clinically detectable disease with normalization of anypreviously abnormal radiographic studies, bone marrow, and cerebrospinalfluid (CSF). Such a response must persist for at least one monthfollowing treatment according to the methods of the invention.Alternatively, an improvement in the disease may be categorized as beinga partial response. By “partial response” is intended at least about a50% decrease in all measurable tumor burden (i.e., the number of tumorcells present in the subject) in the absence of new lesions andpersisting for at least one month. Such a response is applicable tomeasurable tumors only. In addition to these positive therapeuticresponses, the subject undergoing therapy with the antagonist anti-CD40antibody or antigen-binding fragment thereof may experience thebeneficial effect of an improvement in the symptoms associated with thedisease. Thus the subject may experience a decrease in the so-called Bsymptoms, i.e., night sweats, fever, weight loss, and/or urticaria.

[0055] By “therapeutically effective dose or amount” is intended anamount of antagonist anti-CD40 antibody or antigen-binding fragmentthereof that, when administered brings about a positive therapeuticresponse with respect to treatment of a patient with a diseasecomprising malignant B cells. Administration of the pharmaceuticalcomposition comprising the therapeutically effective dose or amount canbe achieved using any acceptable administration method known in the art.Preferably the pharmaceutical composition comprising the antagonistanti-CD40 antibody or antigen-binding fragment thereof is administeredintravenously, preferably by infusion over a period of about 1 to about10 hours, more preferably over about 1 to about 8 hours, even morepreferably over about 2 to about 7 hours, still more preferably overabout 4 to about 6 hours, depending upon the anti-CD40 antibody beingadministered. The initial infusion with the pharmaceutical compositionmay be given over a period of about 4 to about 6 hours with subsequentinfusions delivered more quickly. Subsequent infusions may beadministered over a period of about 1 to about 6 hours, preferably about1 to about 4 hours, more preferably about 1 to about 3 hours, yet morepreferably about 1 to about 2 hours.

[0056] A pharmaceutical composition of the invention is formulated to becompatible with its intended route of administration. Examples of routesof administration include parenteral, e.g., intravenous, intradermal,subcutaneous, oral (e.g., inhalation), transdermal (topical),transmucosal, and rectal administration. Solutions or suspensions usedfor parenteral, intradermal, or subcutaneous application can include thefollowing components: a sterile diluent such as water for injection,saline solution, fixed oils, polyethylene glycols, glycerin, propyleneglycol or other synthetic solvents; antibacterial agents such as benzylalcohol or methyl parabens; antioxidants such as ascorbic acid or sodiumbisulfite; chelating agents such as ethylenediaminetetraacetic acid;buffers such as acetates, citrates or phosphates and agents for theadjustment of tonicity such as sodium chloride or dextrose. pH can beadjusted with acids or bases, such as hydrochloric acid or sodiumhydroxide. The parenteral preparation can be enclosed in ampoules,disposable syringes, or multiple dose vials made of glass or plastic.

[0057] The anti-CD40 antibodies are typically provided by standardtechnique within a pharmaceutically acceptable buffer, for example,sterile saline, sterile buffered water, propylene glycol, combinationsof the foregoing, etc. Methods for preparing parenterally administrableagents are described in Remington 's Pharmaceutical Sciences (18^(th)ed.; Mack Publishing Company, Eaton, Pa. 1990), herein incorporated byreference. See also, for example, WO 98/56418, which describesstabilized antibody pharmaceutical formulations suitable for use in themethods of the present invention.

[0058] The amount of at least one anti-CD40 antibody or fragment thereofto be administered is readily determined by one of ordinary skill in theart without undue experimentation. Factors influencing the mode ofadministration and the respective amount of at least one antagonistanti-CD40 antibody (or fragment thereof) include, but are not limitedto, the particular lymphoma undergoing therapy, the severity of thedisease, the history of the disease, and the age, height, weight,health, and physical condition of the individual undergoing therapy.Similarly, the amount of antagonist anti-CD40 antibody or fragmentthereof to be administered will be dependent upon the mode ofadministration and whether the subject will undergo a single dose ormultiple doses of this anti-tumor agent. Generally, a higher dosage ofanti-CD40 antibody or fragment thereof is preferred with increasingweight of the patient undergoing therapy. The dose of anti-CD40 antibodyor fragment thereof to be administered is in the range from about 0.003mg/kg to about 50 mg/kg, preferably in the range of 0.01 mg/kg to about40 mg/kg. Thus, for example, the dose can be 0.01 mg/kg, 0.03 mg/kg, 0.1mg/kg, 0.3 mg/kg, 0.5 mg/kg, I mg/kg, 1.5 mg/kg, 2 mg/kg, 2.5 mg/kg, 3mg/kg, 5 mg/kg, 7 mg/kg, 10 mg/kg, 15 mg/kg, 20 mg/kg, 25 mg/kg, 30mg/kg, 35 mg/kg, 40 mg/kg, 45 mg/kg, or 50 mg/kg.

[0059] In another embodiment of the invention, the method comprisesadministration of multiple doses of antagonist anti-CD40 antibody orfragment thereof. The method may comprise administration of 1, 2, 3, 4,5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, or more therapeuticallyeffective doses of a pharmaceutical composition comprising an antagonistanti-CD40 antibody or fragment thereof. The frequency and duration ofadministration of multiple doses of the pharmaceutical compositionscomprising anti-CD40 antibody or fragment thereof can be readilydetermined by one of skill in the art without undue experimentation.Moreover, treatment of a subject with a therapeutically effective amountof an antibody can include a single treatment or, preferably, caninclude a series of treatments. In a preferred example, a subject istreated with antagonist anti-CD40 antibody or antigen-binding fragmentthereof in the range of between about 0.1 to 20 mg/kg body weight, onceper week for between about 1 to 10 weeks, preferably between about 2 to8 weeks, more preferably between about 3 to 7 weeks, and even morepreferably for about 4, 5, or 6 weeks. Treatment may occur annually toprevent relapse or upon indication of relapse. It will also beappreciated that the effective dosage of antibody or antigen-bindingfragment thereof used for treatment may increase or decrease over thecourse of a particular treatment. Changes in dosage may result andbecome apparent from the results of diagnostic assays as describedherein. Thus, in one embodiment, the dosing regimen includes a firstadministration of a therapeutically effective dose of at least oneanti-CD40 antibody or fragment thereof on days 1, 7, 14, and 21 of atreatment period. In another embodiment, the dosing regimen includes afirst administration of a therapeutically effective dose of at least oneanti-CD40 antibody or fragment thereof on days 1, 2, 3, 4, 5, 6, and 7of a week in a treatment period. Further embodiments include a dosingregimen having a first administration of a therapeutically effectivedose of at least one anti-CD40 antibody or fragment thereof on days 1,3, 5, and 7 of a week in a treatment period; a dosing regimen includinga first administration of a therapeutically effective dose of at leastone anti-CD40 antibody or fragment thereof on days 1 and 3 of a week ina treatment period; and a preferred dosing regimen including a firstadministration of a therapeutically effective dose of at least oneanti-CD40 antibody or fragment thereof on day 1 of a week in a treatmentperiod. The treatment period may comprise 1 week, 2 weeks, 3 weeks, amonth, 3 months, 6 months, or a year. Treatment periods may besubsequent or separated from each other by a day, a week, 2 weeks, amonth, 3 months, 6 months, or a year.

[0060] The antagonist anti-CD40 antibodies present in the pharmaceuticalcompositions described herein for use in the methods of the inventionmay be native or obtained by recombinant techniques, and may be from anysource, including mammalian sources such as, e.g., mouse, rat, rabbit,primate, pig, and human Preferably such polypeptides are derived from ahuman source, and more preferably are recombinant, human proteins fromhybridoma cell lines.

[0061] The pharmaceutical compositions useful in the methods of theinvention may comprise biologically active variants of the antagonistanti-CD40 antibodies of the invention. Such variants should retain thedesired biological activity of the native polypeptide such that thepharmaceutical composition comprising the variant polypeptide has thesame therapeutic effect as the pharmaceutical composition comprising thenative polypeptide when administered to a subject. That is, the variantanti-CD40 antibody will serve as a therapeutically active component inthe pharmaceutical composition in a manner similar to that observed forthe native antagonist antibody, for example 15B8 as expressed by thehybridoma cell line 15B8. Methods are available in the art fordetermining whether a variant anti-CD40 antibody retains the desiredbiological activity, and hence serves as a therapeutically activecomponent in the pharmaceutical composition. Biological activity ofantibody variants can be measured using assays specifically designed formeasuring activity of the native antagonist antibody, including assaysdescribed in the present invention.

[0062] Suitable biologically active variants of native or naturallyoccurring antagonist anti-CD40 antibodies can be fragments, analogues,and derivatives of that polypeptide. By “fragment” is intended apolypeptide consisting of only a part of the intact polypeptide sequenceand structure, as noted elsewhere herein. By “analogue” is intended ananalogue of either the native polypeptide or of a fragment of the nativepolypeptide, where the analogue comprises a native polypeptide sequenceand structure having one or more amino acid substitutions, insertions,or deletions. By “derivative” is intended any suitable modification ofthe native polypeptide of interest, of a fragment of the nativepolypeptide, or of their respective analogues, such as glycosylation,phosphorylation, polymer conjugation (such as with polyethylene glycol),or other addition of foreign moieties, so long as the desired biologicalactivity of the native polypeptide is retained. Methods for makingpolypeptide fragments, analogues, and derivatives are generallyavailable in the art.

[0063] For example, amino acid sequence variants of an antagonistanti-CD40 antibody can be prepared by mutations in the cloned DNAsequence encoding the antibody of interest. Methods for mutagenesis andnucleotide sequence alterations are well known in the art. See, forexample, Walker and Gaastra, eds. (1983) Techniques in Molecular Biology(MacMillan Publishing Company, New York); Kunkel (1985) Proc. Natl.Acad. Sci. USA 82:488492; Kunkel et al. (1987) Methods Enzymol.154:367-382; Sambrook et al. (1989) Molecular Cloning: A LaboratoryManual (Cold Spring Harbor, N.Y.); U.S. Pat. No. 4,873,192; and thereferences cited therein; herein incorporated by reference. Guidance asto appropriate amino acid substitutions that do not affect biologicalactivity of the polypeptide of interest may be found in the model ofDayhoff et al. (1978) in Atlas of Protein Sequence and Structure (Natl.Biomed. Res. Found., Washington, D.C.), herein incorporated byreference. Conservative substitutions, such as exchanging one amino acidwith another having similar properties, may be preferred. Examples ofconservative substitutions include, but are not limited to, Gly⇄Ala,Val⇄Ile⇄Leu, Asp⇄Glu, Lys⇄Arg, Asn⇄Gln, and Phe⇄Trp⇄Tyr.

[0064] In constructing variants of the antagonist anti-CD40 antibodypolypeptide of interest, modifications are made such that variantscontinue to possess the desired activity, i.e., similar binding affinityand having the following characteristics: 1) are capable of specificallybinding to a human CD40 antigen expressed on the surface of a humancell; 2) are free of significant agonist activity when bound to a CD40antigen on a normal human B cell; and, 3) exhibit antagonist activitywhen bound to a CD40 antigen on a malignant human B cell. Obviously, anymutations made in the DNA encoding the variant polypeptide must notplace the sequence out of reading frame and preferably will not createcomplementary regions that could produce secondary mRNA structure. SeeEP Patent Application Publication No. 75,444.

[0065] Biologically active variants of anti-CD40 antibodies willgenerally have at least 70%, preferably at least 80%, more preferablyabout 90% to 95% or more, and most preferably about 98% or more aminoacid sequence identity to the amino acid sequence of the referencepolypeptide molecule, which serves as the basis for comparison. Abiologically active variant of a reference antagonist anti-CD40 antibodyhaving the specificity and binding characteristics described herein maydiffer from the reference polypeptide by as few as 1-15 amino acids, asfew as 1-10, such as 6-10, as few as 5, as few as 4, 3, 2, or even 1amino acid residue. By “sequence identity” is intended the same aminoacid residues are found within the variant polypeptide and thepolypeptide molecule that serves as a reference when a specified,contiguous segment of the amino acid sequence of the variant is alignedand compared to the amino acid sequence of the reference molecule. Thepercentage sequence identity between two amino acid sequences iscalculated by determining the number of positions at which the identicalamino acid residue occurs in both sequences to yield the number ofmatched positions, dividing the number of matched positions by the totalnumber of positions in the segment undergoing comparison to thereference molecule, and multiplying the result by 100 to yield thepercentage of sequence identity.

[0066] For purposes of optimal alignment of the two sequences, thecontiguous segment of the amino acid sequence of the variants may haveadditional amino acid residues or deleted amino acid residues withrespect to the amino acid sequence of the reference molecule. Thecontiguous segment used for comparison to the reference amino acidsequence will comprise at least twenty (20) contiguous amino acidresidues, and may be 30, 40, 50, 100, or more residues. Corrections forincreased sequence identity associated with inclusion of gaps in thevariant's amino acid sequence can be made by assigning gap penalties.Methods of sequence alignment are well known in the art for both aminoacid sequences and for the nucleotide sequences encoding amino acidsequences.

[0067] Thus, the determination of percent identity between any twosequences can be accomplished using a mathematical algorithm. Onepreferred, non-limiting example of a mathematical algorithm utilized forthe comparison of sequences is the algorithm of Myers and Miller (1988)CABIOS4:11-17. Such an algorithm is utilized in the ALIGN program(version 2.0), which is part of the GCG sequence alignment softwarepackage. A PAM120 weight residue table, a gap length penalty of 12, anda gap penalty of 4 can be used with the ALIGN program when comparingamino acid sequences. Another preferred, nonlimiting example of amathematical algorithm for use in comparing two sequences is thealgorithm of Karlin and Altschul (1990) Proc. Natl. Acad. Sci. USA87:2264, modified as in Karlin and Altschul (1993) Proc. Natl. Acad.Sci. USA 90:5873-5877. Such an algorithm is incorporated into the NBLASTand XBLAST programs of Altschul et al. (1990) J. Mol. Biol. 215:403.BLAST nucleotide searches can be performed with the NBLAST program,score=100, wordlength=12, to obtain nucleotide sequences homologous to anucleotide sequence encoding the polypeptide of interest. BLAST proteinsearches can be performed with the XBLAST program, score=50,wordlength=3, to obtain amino acid sequences homologous to thepolypeptide of interest. To obtain gapped alignments for comparisonpurposes, Gapped BLAST can be utilized as described in Altschul et al.(1997) Nucleic Acids-Res. 25:3389. Alternatively, PSI-Blast can be usedto perform an iterated search that detects distant relationships betweenmolecules. See Altschul et al. (1997) supra. When utilizing BLAST,Gapped BLAST, and PSI-Blast programs, the default parameters of therespective programs (e.g., XBLAST and NBLAST) can be used. Seehttp://www.ncbi.nlm.nih.gov. Also see the ALIGN program (Dayhoff (1978)in Atlas of Protein Sequence and Structure 5:Suppl. 3 (NationalBiomedical Research Foundation, Washington, D.C.) and programs in theWisconsin Sequence Analysis Package, Version 8 (available from GeneticsComputer Group, Madison, Wis.), for example, the GAP program, wheredefault parameters of the programs are utilized.

[0068] When considering percentage of amino acid sequence identity, someamino acid residue positions may differ as a result of conservativeamino acid substitutions, which do not affect properties of proteinfunction. In these instances, percent sequence identity may be adjustedupwards to account for the similarity in conservatively substitutedamino acids. Such adjustments are well known in the art. See, forexample, Myers and Miller (1988) Computer Applic. Biol. Sci. 4:11-17.

[0069] The precise chemical structure of a polypeptide capable ofspecifically binding CD40 and retaining antagonist activity,particularly when bound to CD40 antigen on malignant B cells, depends ona number of factors. As ionizable amino and carboxyl groups are presentin the molecule, a particular polypeptide may be obtained as an acidicor basic salt, or in neutral form. All such preparations that retaintheir biological activity when placed in suitable environmentalconditions are included in the definition of antagonist anti-CD40antibodies as used herein. Further, the primary amino acid sequence ofthe polypeptide may be augmented by derivatization using sugar moieties(glycosylation) or by other supplementary molecules such as lipids,phosphate, acetyl groups and the like. It may also be augmented byconjugation with saccharides. Certain aspects of such augmentation areaccomplished through post-translational processing systems of theproducing host; other such modifications may be introduced in vitro. Inany event, such modifications are included in the definition of ananti-CD40 antibody used herein so long as the antagonist properties ofthe anti-CD40 antibody are not destroyed. It is expected that suchmodifications may quantitatively or qualitatively affect the activity,either by enhancing or diminishing the activity of the polypeptide, inthe various assays. Further, individual amino acid residues in the chainmay be modified by oxidation, reduction, or other derivatization, andthe polypeptide may be cleaved to obtain fragments that retain activity.Such alterations that do not destroy antagonist activity do not removethe polypeptide sequence from the definition of anti-CD40 antibodies ofinterest as used herein.

[0070] The art provides substantial guidance regarding the preparationand use of polypeptide variants. In preparing the anti-CD40 antibodyvariants, one of skill in the art can readily determine whichmodifications to the native protein nucleotide or amino acid sequencewill result in a variant that is suitable for use as a therapeuticallyactive component of a pharmaceutical composition used in the methods ofthe present invention.

[0071] Any pharmaceutical composition comprising an antagonist anti-CD40antibody as the therapeutically active component can be used in themethods of the invention. Thus liquid, lyophilized, or spray-driedcompositions comprising antagonist anti-CD40 antibodies or variantsthereof that are known in the art may be prepared as an aqueous ornonaqueous solution or suspension for subsequent administration to asubject in accordance with the methods of the invention. Each of thesecompositions will comprise anti-CD40 antibodies or variants thereof as atherapeutically or prophylactically active component. By“therapeutically or prophylactically active component” is intended theanti-CD40 antibody or variant thereof is specifically incorporated intothe composition to bring about a desired therapeutic or prophylacticresponse with regard to treatment, prevention, or diagnosis of a diseaseor condition within a subject when the pharmaceutical composition isadministered to that subject. Preferably the pharmaceutical compositionscomprise appropriate stabilizing agents, bulking agents, or both tominimize problems associated with loss of protein stability andbiological activity during preparation and storage.

[0072] Formulants may be added to pharmaceutical compositions comprisingan anti-CD40 antibody of the invention. These formulants may include,but are not limited to, oils, polymers, vitamins, carbohydrates, amineacids, salts, buffers, albumin, surfactants, or bulking agents.Preferably carbohydrates include sugar or sugar alcohols such as mono-,di-, or polysaccharides, or water soluble glucans. The saccharin orglucans can include fructose, glucose, mannose, sorbose, xylose,maltose, sucrose, dextran, pullulan, dextrin, αand β, cyclodextrin,soluble starch, hydroxyethyl starch, and carboxymethylcellulose, ormixtures thereof. “Sugar alcohol” is defined as a C₄ to C₈ hydrocarbonhaving a hydroxyl group and includes galactitol, inositol, mannitol,xylitol, sorbitol, glycerol, and arabitol. These sugars or sugaralcohols may be used individually or in combination. The sugar or sugaralcohol concentration is between 1.0% and 7% w/v., more preferablybetween 2.0% and 6.0% w/v. Preferably amino acids include levorotary (L)forms of carnitine, arginine, and betaine; however, other amino acidsmay be added. Preferred polymers include polyvinylpyrrolidone (PVP) withan average molecular weight between 2,000 and 3,000, or polyethyleneglycol (PEG) with an average molecular weight between 3,000 and 5,000.Surfactants that can be added to the formulation are shown in EP Nos.270,799 and 268,110.

[0073] Additionally, antibodies can be chemically modified by covalentconjugation to a polymer to increase their circulating half-life, forexample. Preferred polymers, and methods to attach them to peptides, areshown in U.S. Pat. Nos. 4,766,106; 4,179,337; 4,495,285; and 4,609,546;which are all hereby incorporated by reference in their entireties.Preferred polymers are polyoxyethylated polyols and polyethylene glycol(PEG). PEG is soluble in water at room temperature and has the generalformula: R(O—CH₂—CH₂)_(n) O—R where R can be hydrogen, or a protectivegroup such as an alkyl or alkanol group. Preferably, the protectivegroup has between 1 and 8 carbons, more preferably it is methyl. Thesymbol n is a positive integer, preferably between 1 and 1,000, morepreferably between 2 and 500. The PEG has a preferred average molecularweight between 1,000 and 40,000, more preferably between 2,000 and20,000, most preferably between 3,000 and 12,000. Preferably, PEG has atleast one hydroxy group, more preferably it is a terminal hydroxy group.It is this hydroxy group which is preferably activated to react with afree amino group on the inhibitor.

molecular weight in the same range as PEG The structure for POG is shownin Knauf et al. (1988) J. Bio. Chem. 263:15064-15070, and a discussionof POG/IL-2 conjugates is found in U.S. Pat. No. 4,766,106, both ofwhich are hereby incorporated by reference in their entireties.

[0074] Another drug delivery system for increasing circulatory half-lifeis the liposome. Methods of preparing liposome delivery systems arediscussed in Gabizon et al. (1982) Cancer Research 42:4734; Cafiso(1981) Biochern Biophys Acta 649:129; and Szoka (1980) Ann. Rev.Biophys. Eng. 9:467. Other drug delivery systems are known in the artand are described in, e.g., Poznansky et al. (1980) Drug DeliverySystems (R.L. Juliano, ed., Oxford, N.Y.) pp. 253-315; Poznansky (1984)Pharm Revs 36:277.

[0075] A further embodiment of the invention is the use of antagonistanti-CD40 antibodies for diagnostic monitoring of protein levels intissue as part of a clinical testing procedure, e.g., to determine theefficacy of a given treatment regimen. Detection can be facilitated bycoupling the antibody to a detectable substance. Examples of detectablesubstances include various enzymes, prosthetic groups, fluorescentmaterials, luminescent materials, bioluminescent materials, andradioactive materials. Examples of suitable enzymes include horseradishperoxidase, alkaline phosphatase, β-galactosidase, oracetylcholinesterase; examples of suitable prosthetic group complexesinclude streptavidin/biotin and avidin/biotin; examples of suitablefluorescent materials include umbelliferone, fluorescein, fluoresceinisothiocyanate, rhodamine, dichlorotriazinylamine fluorescein, dansylchloride or phycoerythrin; an example of a luminescent material includesluminol; examples of bioluminescent materials include luciferase,luciferin, and aequorin; and examples of suitable radioactive materialinclude ¹²⁵I ¹³¹I ³⁵S, or ³H.

[0076] The antagonist anti-CD40 antibodies can be used in combinationwith known chemotherapeutics and cytokines for the treatment of diseasestates comprising malignant B cells. For example, the anti-CD40antibodies of the invention can be used in combination with cytokinessuch as interleukin-2. In another embodiment, the anti-CD40 antibodiesof the invention can be used in combination with Rituximab (IDEC-C2B8;Rituxan®; IDEC Pharmaceuticals Corp., San Diego, Calif.). Rituximab is achimeric anti-CD20 monoclonal antibody containing human IgG1 and kappaconstant regions with murine variable regions isolated from a murineanti-CD20 monoclonal antibody, IDEC-2B8 (Reff et al. (1994) Blood83:435-445).

[0077] The anti-CD40 antibodies described herein can further be used toprovide reagents, e.g., labeled or labelable antibodies that can beused, for example, to identify cells expressing CD40. This can be veryuseful in determining the cell type of an unknown sample. Panels ofmonoclonal antibodies can be used to identify tissue by species and/orby organ type. In a similar fashion, these anti-CD40 antibodies can beused to screen tissue culture cells for contamination (i.e., screen forthe presence of a mixture of CD40-expressing and non-CD40 expressingcells in a culture).

[0078] The following examples are offered by way of illustration and notby way of limitations

EXPERIMENTAL

[0079] The antagonist anti-CD40 antibody used in the examples below is15B8. 15B8 is a human IgG₂ subtype anti-human CD40 monoclonal antibodygenerated by immunization of transgenic mice bearing the human IgG2heavy chain locus and the human K light chain locus (Xenomouse,Abgenix). As shown by FACS analysis, 15B8 binds specifically to humanCD40 and cross-reacts with CD40 expressed on the peripheral blood Bcells from monkeys (cynomologus, rhesus and baboons) and chimpanzees.15B8 does not cross-react with CD40 from non-primate animal species, nordoes it bind to other members of the TNF receptor family as demonstratedby ELISA and FACS analysis. The binding affinity of 15B8 to human CD40is 3.1×10⁻⁹ M as determined by BIACore assay.

EXAMPLE 1 Effect of 15B8 on the CD40/CD40L Interaction In Vitro

[0080] A competitive binding assay was performed to determine if directcompetition for CD40 binding is a mechanism of the antagonist activityof 15B8.

[0081] A line of Chinese Hamster Ovary (CHO) cells containing the geneencoding CD40L and expressing CD40L on the cell surface was generated.The CD40L-expressing CHO cells were incubated with purified CD40 beforeand after incubation of CD40 with 15B8. Fluorescein isothiocyanate(FITC)-labeled anti-huIgG was added to the cells. FACS analysis wasperformed to detect 15B8 bound to the CHO cells via CD40. The binding of15B8 to CD40 inhibited the subsequent binding of CD40L to CD40. However,when CD40L and CD40 were incubated together prior to the addition of15B8, 15B8 was subsequently able to bind CD40. While not bound by anymechanism of action, this suggests that 15B8 does not compete directlywith CD40L for binding sites on CD40, and that the binding of 15B8 toCD40 possibly caused conformational changes in the CD40 molecule thatprevented the binding of CD40L to CD40. The putative structuralalteration of the CD40 molecule induced by 15B8 binding could alsodeliver a negative signal to the cell causing the antagonist effect.

EXAMPLE 2 Pharmacologic Action of 15B8 in Lymphoma Cells from NHLPatients

[0082] To demonstrate the potential efficacy of 15B8 in a preclinical invitro model of non-Hodgkin's lymphoma (NHL), 15B8 was tested usingmalignant B cells (NHL cells) obtained from NHL patients who were eitherRituximab-treated or naïve. Rituxinab (IDEC-C2B8; Rituxan®; IDECPharmaceuticals Corp., San Diego, Calif.) is an anti-CD20 monoclonalantibody for the treatment of relapsed or refractory low-grade orfollicular NHL.

[0083] Since primary lymphoma cells do not proliferate in regularculture medium and undergo apoptosis after a few days in culture, tumorcells were co-cultured with irradiated CD40-ligand (CD40L) transfectedfeeder cells (Arpin et al. (1995) Science 268:720-722) in the presenceor absence of the B-cell growth factor interleukin-4 (IL-4). Antibodies(agonist anti-CD40 MS81, antagonist anti-CD40 15B8, or isotype controlhuman IgG2 (huIgG2)) of indicated concentration (from 0.01 μg/ml to 10μg/ml) were then added to the culture. Following incubation at 37° C.for 48 hours, cultured cells were pulsed with ³H-thymidine for 18 hours.The cells were then harvested and analyzed for the amount of³H-thymidine incorporation (Schultze et al. (1995) Proc. Natl. Acad.Sci. USA 92:8200-8204). All sample conditions were in triplicate.

[0084] In these NHL cell primary culture assays, 15B8 alone or incombination with IL-4 did not stimulate NHL cells to proliferate invitro. In contrast, an agonist anti-CD40 MS81 induced NHL cellproliferation under the same conditions. 15B8 showed statisticallysignificant inhibition of NHL cell proliferation stimulated by CD40L(P=0.05) and by CD40L plus IL-4 (P<0.05) in vitro. At 1-10 μg/ml or0.1-10 μg/ml concentration range respectively, 15B8 showed astatistically significant dose-related inhibition of NHLcell-proliferation stimulated by CD40L or by CD40L plus IL-4(P<0.005)(data not shown).

[0085] There are two types of preclinical models that are currently usedfor evaluation of human antigen-specific monoclonal antibodies (Mabs) intherapeutic development for lymphomas. One model is the xenograft mousein vivo model, where the EBV-transformed lymphoma cell lines, such asDaudi (Burlitt lymphoma) or Raji (Burlitt lymphoma) cells, arexenografted into SCID/Nude mice. The defects of these models are thatthe results only reflect effects on the particular immortal cell line,which is derived from one EBV-transformed cell. It is known that Burkittlymphoma cells are lymphoblastoid cells (Ambinder et al. (1999) CancerTreat. Res. 99:2745; Quintanilla-Martinez et al. (1998) Leuk Lymphoma30:111-121; Klein (1996) Acta Microbiol. Immunol. Hung. 43:97-105) whilethe lymphoma cells from NHL patients are believed to be at the mature Bcell stage (Ghia et al. (2000) Adv. Cancer Res. 79:157-173). EBVtransformation of B cells results in changes of many components in theCD40 signaling pathway (Uchida et al. (1999) Science 286:300-303;Farrell et al. (1997) Biomed. Pharmacother. 51:258-267). In contrast toCD40 signaling in NHL cells and normal B cells, CD40 signaling leads togrowth arrest in EBV-transformed Burkitt lymphoma cell lines (Fukuda etal. (2000) Viral Immunol. 13:215-229; Baker et al. (1998) Blood92:2830-2843). Thus, the results of testing an antagonist anti-CD40 MAb(15B8) in the xenograft models will not be able to predict the responseto the antibody (15B8) by NHL patients.

[0086] The other model is the in vitro growth inhibition assay oflymphoma cells from NHL patients, which was used above. The advantage isthat the results predicate the sensitivity of the lymphoma cells fromNHL patients to the agent (15B8) tested. However, the results areobtained from in vitro study under defined conditions. A previouspublished study reported that a rat anti-mouse CD40, which failed toinduce ADCC and CDC in vitro, showed good efficacy in two syngeneicmouse B lymphoma models (BCL1 and A31) (Tutt et al. (1998) J. Immunol.161:3176-3185). The anti-tumor effect of the anti-mouse CD40 occurredslower in time than an anti-Id tested. One of the hypotheses was thatthe anti-mouse CD40 operated by blocking critical growth signals thatare dependent on the expression of surface CD40, not direct signalinglike anti-Id in the mouse models tested. When tested, 15B8 did not bindto the Fcγ receptors in vitro and failed to induce ADCC and CDC in vitro(data not shown) since it is of human IgG2 subtype. 15B8 has similarproperties to the rat anti-mouse CD40. These data support the hypothesisthat 15B8 will be beneficial to NHL patients, especiallyRituxan®-resistant patients.

EXAMPLE 3 Effect of 15B8 on Malignant B-Cell Proliferation In Vitro

[0087] To test if 15B8 provides the growth signal like CD40L in vitro, Bcells from tumor infiltrated lymph nodes (NHL cells) were obtained fromone antibody naïve, one Rituximab-sensitive and one Rituximab-resistantNHL patient. The NHL cells were studied under four different cultureconditions: no added antibody (medium); addition of human isotypeantibody IgG2 (control; referred to as huIgG2); addition of anti-CD40antibody MS81 (agonistic antibody); and addition of 15B8. All antibodieswere tested at 1, 2, and 5 μg/ml in the presence or absence of IL-4. TheNHL cells from two patients were cultured as described above under thesame four conditions in the presence of IL-4 (2 ng/ml). B-cellproliferation was measured by ³H-thymidine incorporation as describedabove.

[0088] Anti-CD40 antibody 15B8, at concentrations of 1, 2, and 5 μg/ml,did not stimulate NHL cells to proliferate in either the absence orpresence of IL-4. In contrast, an agonistic anti-CD40 antibody (MS81),tested at the same concentration, stimulated NHL-cell proliferation bothin the presence and absence of IL-4 in all patient samples.Representative results from one patient are shown in FIGS. 1 and 2.Results from the NHL cells from the two patients in the presence of IL-4and three patients in the absence of IL-4 were comparable. These resultsindicate that 15B8 is not an agonist anti-CD40 antibody and does notstimulate proliferation of NHL cells from Rituximab-sensitive, naïve orRituximab-resistant NHL patients in vitro.

[0089] FACS analysis of the NHL cells was performed with either adirect-labeled 15B8-FITC or 15B8 plus anti-huIgG2-FlTC to confirm thatCD40 is expressed on the surface the NHL cells tested and that 15B8binds to the NHL cells. The NHL cells from 2 Rituximab-sensitive and 4Rituximab-resistant patients (6 patients in total) were tested. NHLcells from all the patients expressed CD40 and bound 15B8. The 15B8binding-positive cell population in any given patient was about 66% to91%.

EXAMPLE 4 15B8 Inhibits CD40L-Stimulated Proliferation of NHL Cells InVitro

[0090] To evaluate the ability of 15B8 to block the growth signalprovided by CD40L in vitro, NHL cells from patients were cultured asdescribed above in suspension over CD40L-expressing feeder cells underfour different conditions: no added antibody (medium); addition of humanisotype antibody IgG2 (control); addition of anti-CD40 antibody MS81(agonistic antibody); and addition of 15B8. All antibodies were added atconcentrations of 1, 2, and 5 μg/ml in the presence or the absence ofIL-4. The NHL cells from 1 antibody-naïve, 2 Rituximab-sensitive, and 5Rituximab-resistant patients (8 patients in total) were cultured underthe same four conditions as described above in the presence of IL-4 (2ng/ml). NHL cells from 3 Rituximab-sensitive and 4 Rituximab-resistantpatients (7 patients in total) were cultured under similar conditions inthe absence of IL-4. The NHL cell proliferation was measured by³H-thymidine incorporation.

[0091] Table 1 below shows the inhibitory effect of 15B8 on theproliferation of NHL cells from 2 Rituximab-sensitive (data from onepatient reproducible in two separate experiments) and 4Rituximab-resistant patients (6 patients in total) stimulated by CD40Lalone in vitro. Representative results from the cells of one patient (A)are shown in FIG. 3. 15B8 inhibited the proliferation by about 12-68%when compared to the control in the 6 patients. The degree of inhibitionby 15B8 varied depending on patient samples and the dose level of 15B8.Statistical analysis of the data from 6 of the 7 patient samples testedshows that the inhibition of CD40L-stimulated NHL cell proliferation by15B8 is significant at 1 μg/ml (p=0.05). There is a statisticallysignificant dose response (p<0.005), as the inhibitory effect increaseswith increasing 15B8 dose. TABLE 1 Effect of 15B8 MAb on CD40-Lstimulation of proliferation of NHL patient cells in the absence ofIL-4.¹ Treatment Dose 15B8 Patient ID Patient Type² (μg/ml) %Inhibition³ A CR 1 56.61 2 58.99 5 63.16 A CR 1 61.96 2 60.41 5 64.75 1060.29 B CR 1 None 2 None 5 None 10 12.11 D NR 1 52.22 2 61.63 5 68.04 1068.17 E NR 1 13.07 2 22.34 5 31.04 10 31.87 F NR 1 24.51 2 27.43 5 38.7110 47.35 G NR 1 11.12 2 22.41 5 30.61 10 43.15

[0092] Table 2 (below) shows the inhibitory effect of 15B8 onproliferation of NHL cells from 1 antibody-naïve, 2 Rituximab-sensitive(data from both patient samples were repeated twice reproducibly), and 5Rituximab-resistant patients (8 patients in total) stimulated by bothCD40L and IL-4 in vitro. At 1 μg/ml level, 15B8 significantly (p<0.005)inhibited the CD40L and IL-4 mediated proliferation of the NHL cells.The degree of inhibition ranged from 18-69% at high dose (5 or 10 μg/ml)in samples from all 8 patients in vitro. There was a statisticallysignificant dose response of this inhibitory effect by 15B8 (p<0.005) ata 15B8 concentration range of 0.01-10 μg/ml. FIG. 4 shows onerepresentative dose response curve. These in vitro results suggest thattreatment with 15B8 may block the CD40-mediated growth signal for NHLcells in patients. TABLE 2 Effect of 15B8 Mab on CD40-L stimulation ofNHL patient cells in the presence of IL-4.¹ Treatment Dose 15B8 PatientID Patient Type² (μg/ml) % Inhibition³ A CR 1 34.39 2 30.54 5 36.42 A CR0.01 0.44 0.04 23.32 0.2 29.54 1 35.38 5 46.12 10 48.63 C CR 1 34.91 240.89 5 56.34 10 69.21 C CR 1 None 2 16.79 5 21.64 10 12.63 D NR 1 1.952 6.43 5 20.95 10 26.31 E NR 1 1.91 2 2.74 5 28.36 10 28.26 E NR 1 None2 11.76 5 27.54 10 34.07 G NR 1 39.38 2 32.74 5 36.48 10 37.78 H NR 1None 2 None 5 7.81 10 18.47 I Naive 0.01 None 0.04 13.16 0.2 15.64 116.20 5 21.53 10 24.51

EXAMPLE 5 15B8 Does Not Activate Human Peripheral Blood B Cells and DoesNot Cause PBMC Proliferation In Vitro in Human, Chimpanzee, and Marmoset

[0093] To determine if it is an agonist or antagonist anti-CD40, 15B8was tested in several in vitro assays described below using cells fromhumans and five different primate species, including chimpanzee (chimp),marmoset, cynomologus monkey, rhesus monkey, and baboon. TABLE 3Stimulation of PBMC/B-cell proliferation in human, chimp, and marmosetby 15B8 antibody.¹ Number of Dose CD40L, Fold 15B8, Fold Species CellSource Samples (μg/ml) huIgG2, Base Increase³ Increase² Human B 2 5 170.58/36.33 1.77/4.37 2 1 1 70.58/36.33  3.1/5.4 2 0.2 1 70.58/36.331.16/4.63 Human PBMC 5 5 1  9.36-91.60 0.49-2.28 15 1 1  9.36-91.600.35-2.38 12 0.2 1  9.36-91.60 0.41-3.74 Marmoset PBMC 3 5 1 29.24-90.32.05-7.2 Monkey 5 1 1  7.99-90.3 1.35-5.79 Chimp PBMC 1 5 1 10.15 2.46 51 1  5.12-9.2 0.66-5.2

[0094] Upon B-cell activation, a number of cell surface proteins areup-regulated (Denton et al. (1998) Pediatr. Transplant. 2:6-15; Evans etal (2000) J. Immunol. 164:688-697; Noelle (1998) Agents Actions Suppl.49:17-22; Lederman et al (1996) Curr. Opin. Hematol. 3:77-86). Toconfirm 15B8 does not activate human B cells and does not induce anagonist signal when bound to CD40, its ability to up-regulate B cellactivation markers was tested by FACS analysis using purified humanPBMC. There was no up-regulation in the expression of activation markerssuch as CD25, CD69, CD86, HLA-DR, and ICAM-1 (CD54) in 15B8 treatedhuman B cells (Table 4). The level of these markers was similar whencells were treated with either 15B8 or huIgG2 control (Table 4). Incontrast, CD69 was consistently up-regulated by CD40L in PBMC samplesfrom 3 healthy volunteers tested. TABLE 4 Effect of 15B8 onup-regulation of B-cell activation markers in vitro by FACS. CellIncubation Number of Species Source Time Subjects CD54 CD69 HLA-DR CD25CD80 CD86 Human CD20 4 h-24 h 3 — — — — N/A — from PBMC Chimp CD20 4h-24 h 3 N/A — N/A N/A N/A N/A from PBMC

[0095] Additional consequences of B cell activation are up-regulation ofsurface FasL and apoptosis (Revy et al. (1998) Eur. J. Immunol.28:3648-3654; Carey et al. (2000) Immunol. Rev. 176:105-115; Ju et al.(1999) Int. Rev. Immunol. 18:485-513; Baumgarth (2000) Immunol. Rev.176:171-180). To confirm 15B8 is not an agonistic anti-CD40 antibody,its ability to induce FasL expression and apoptosis of human B cells wasalso tested. Annexin V staining on the cell surface can be used as anearly apoptosis marker (Ju et al. (1999) Int. Rev. Immutiol.18:485-513). Human B cells were purified from peripheral blood andincubated with 15B8. FACS analysis was used to detect cells withpositive staining of Annexin V and anti-FasL. There was no significantdifference on the surface staining by the two reagents between cellsincubated with 15B8 or the isotype control (huIgG2) antibody (data notshown). This result shows that 15B8 does not induce apoptosis of human Bcells in vitro. These data provide further evidence that 15B8 is not anagonist anti-CD40 antibody for human B cells.

[0096] 15B8 cross-reacts with CD40 expressed on the surface of CD20positive PBMCs from primates. To test if 15B8 can activate CD40 on Bcells from other primate species such as chimpanzees and marmosets, thesame proliferation assays were carried out using freshly isolated chimpand marmoset PBMC from 15 chimps and 5 marmosets. Similar to the resultswith the human PBMC, 15B8 did not stimulate the proliferation in vitroof PBMCs from 6 chimps and 5 marmosets at 1 and 5 μg/ml concentration(Table 3 above). 15B8 also did not up-regulate the expression ofactivation marker, CD69, in the three chimp-PBMC samples tested (Table4). 15B8 did not show any effect on FasL expression and apoptosis inchimp PBMCs similar to human PBMC controls after 24 and 48 hoursstimulation in vitro in all samples from 6 chimps tested (data notshown).

[0097] Cross-linking 15B8 by a secondary antibody fixed to plasticsurface did not increase its potency to stimulate B-cell proliferation(data not shown). When tested using PBMCs from humans and chimps in thiscross-linking assay, 15B8 did not stimulate proliferation of the cells.This observation indicates a reduced risk of 15B8 being stimulative(i.e., agonistic or having agonist activity) for B-cell proliferation incase of induction of anti-15B8 (HAHA) or Fc binding to other Fc receptorexpressing cells when administered in vivo.

[0098] In summary, 15B8 does not initiate an activation signal in humanB cells/PBMCs nor in chimp/marmoset PBMCs in vitro. Therefore, 15B8 isnot an agonist anti-CD40 antibody in human, chimps, and marmosets.

EXAMPLE 6 15B8 is an Antagonist Anti-CD40 Antibody in Humans,Chimpanzees, and Marmosets In Vitro

[0099] To determine if 15B8 is an antagonist anti-CD40, its ability toinhibit CD40-CD40L interaction was tested in a CD40l-mediated humanB-cell proliferation assay (Kwekkeboom et al. (1993) Immunology79:439-444). A transfected CHO cell line expressing human CD40L was usedto stimulate the proliferation of purified human peripheral blood Bcells or PBMCs. Human B cells from 10 healthy volunteers and human PBMCsfrom 3 healthy volunteers were tested. In all the samples tested, 15B8suppressed CD40L-expressing CHO cells mediated-proliferation by 42-88%at concentration range from 0.2-5 μg/ml (Table 5). FIG. 5 shows arepresentative dose-response curve using cells from 3 individuals. Theno-effect dose of 15B8 is 0.008 μg/ml and reaches saturating dose at 0.2μg/ml (FIG. 5). This observation indicates that 15B8, as an antagonistanti-CD40 antibody, can inhibit the growth signals in human B cells andPBMCs provided by cell surface-expressed CD40L. TABLE 5 Inhibition ofCD40L-inducted-proliferation of PBMC/B cell with 15B8 antibody.¹ Numberf Dose HuIgG2, % of 15B8, % of Species Cell Source Samples (μg/ml) CD40L(Base) Inhibition Inhibition² Human B 7 5 100 (−27)-14% 45-85% 9 1 100(−93)-11% 42-87% 6 0.2 100 (−20)-(−6)% 44-82% Human PBMC 1 5 100 13% 45%2 1 100    3-32% 76-88% Marmoset PBMC 3 1 100    1-35% 68-84% MonkeyChimpanzee PBMC 3 1 100  (−3)-21% 55-73%

[0100] Additional assays were carried out using freshly isolated PBMCsfrom 9 chimps and 3 marmosets. As with the human PBMCs, 15B8 was able toinhibit the proliferation of chimp and marmoset PBMCs stimulated byCD40L-expressing-CHO cells at 1 μg/ml concentration level (Table 5above). The inhibition by 15B8 was approximately 55-73% and 68-84% inPBMC samples from 3 chimps and 3 marmosets respectively (Table 5 above).

[0101] Activated B cells undergo a number of biological responses suchas proliferation and antibody production. The activation of B cells by Tcell-dependent antigens involves CD4 ⁺T-helper (Th) cells. This T cellhelper process is mediated by a concerted effort of the interaction ofCD40 on the B cells with the CD40L on the Th cells surface together withthe interactions of other co-stimulatory factors and cytokines (Dentonet al. (1998) Pediatr. Transplant. 2:6-15; Evans et al. (2000) J.Immunol. 164:688-697; Noelle (1998) Agents Actions Suppl. 49:17-22;Lederman et al. (1996) Curr. Opin. Hematol. 3:77-86; Mackey et al.(1998) J. Leukoc. Biol. 63:418428). To test if 15B8 can block T-helpercell mediated B cell antibody production, purified human peripheralblood B cells were cultured in the presence of purified irradiated Tcells activated with anti-CD3 antibody. An ELISA assay was used tomeasure the level of IgM production. 15B8 reduced IgM production byabout 30% in this assay (data not shown). Therefore, 15B8 can reduceT-cell-mediated B-cell immunoglobulin production.

[0102] In summary, 15B8 inhibits CD40L-induced B-cell/PBMC proliferationin human, chimp and marmoset, and inhibits T-cell induced antibodyproduction by purified human B cells in vitro. These data demonstratethat 15B8 is an antagonist anti-CD40 antibody in human B cells and PBMCsfrom chimps and marmosets in vitro.

EXAMPLE 7 15B8 is an Agonist Anti-Monkey (Cynomologus, Rhesus, andBaboon) CD40 Antibody In Vitro

[0103] FACS analysis demonstrates that 15B8 binds to CD40 expressed onthe surface of B cells from peripheral blood of monkeys (rhesus,cynomologus, and baboon). The effect of 15B8 on freshly isolatedcynomologus monkey PBMC was tested in the same proliferation assaydescribed above for human and chimps (Coligan et al. (1998) CurrentProtocols in Immunology 13:12; Kwekkeboom et al. (1993) Immunology79:439444). In contrast to human PBMC, 15B8 was found to stimulatecynomologus monkey PBMC to proliferate in vitro as measured by ³Hmethyl-thymidine incorporation (Table 6 below). At 1 μg/ml level, 15B8stimulated the proliferation of the PBMCs by 6-fold to 129.7-foldcompare to the huIgG2 control in the twenty-two samples from 17 monkeystested (samples from 5 monkeys were tested twice) (Table 6 below). At 5μg/ml level, the proliferation stimulated by 15B8 is 14-fold to 24-foldin four samples from 2 monkeys and about 1.25-fold or 1.85-fold in twosamples from 2 monkeys (Table 6). This suggests that, at concentrationlevel of 5 μg/ml, 15B8 may be at the limit of over-saturating dose forits proliferation-stimulatory effect on PBMCs from cynomologus monkey.Further FACS analysis of B cells for activation status by surfacemarkers indicated that 15B8 induces CD69, CD86, and HLA-DR up-regulationon monkey B cells (Table 7). These data suggest that 15B8 is an agonistantibody to CD40 expressed on peripheral blood B cells from cynomologusmonkeys in vitro.

[0104] To confirm that this agonistic effect of 15B8 is notcynomologus-monkey specific, the same assays were performed using PBMCsfrom rhesus monkeys and baboons. Similar results to that obtained fromcells of cynomologus monkeys were observed as shown in Table 6. 15B8stimulated proliferation of PBMCs from rhesus monkeys and baboons invitro (Table 6). The agonist activity of 15B8 is shown using the PBMCsfrom 5 rhesus monkeys and 3 baboons (Table 6). TABLE 6 Proliferation ofPBMCs from human, cynomologus and rhesus monkeys, and baboons stimulatedby 15B8.¹ Number of CD40L, Fold 15B8, Fold Species Cell Source SamplesDose (ug/ml) huIgG2, Base Increase³ Increase² Human PBMC 5 5 1 9.36-91.60  0.49-2.28 15 1 1  9.36-91.60  0.35-2.38 12 0.2 1 9.36-91.60  0.41-3.74 Rhesus PBMC 5 1 1 12.71-89.67 27.34-50.9 MonkeyCyno PBMC 6 5 1 14.57-124.01  1.25-24.53 Monkey 22 1 1  5.15-167.73 6.13-129.74 3 0.2 1 77.01-124.01  0.9-67.56 Baboon PBMC 3 1 1 5.19-175.07  3.32-113.28

[0105] TABLE 7 Effect of 15B8 on unregulation of B-cell activationmarkers in vitro by FACS analysis. Cell incubation Number of SpeciesSource time subjects CD54 CD69 HLA-DR CD25 CD80 CD86 Human CD20 4 h-24 h3 — — — — N/A — from PBMC Cyno CD20/1 4 h-3 day 2 N/A 1/2 up 1.1 up — —1/1 up Monkey 9 from (day 3) (day 3) PBMC

EXAMPLE 8 15B8 is an Agonist Anti-CD40 Antibody In Vivo in CynomologusMonkeys

[0106] 15B8 can stimulate proliferation and up-regulation of cellsurface activation markers in PBMCs from cynomologus monkeys in vitro.To determine if 15B8 is an agonist anti-CD40 antibody in these monkeysin vivo, a study was performed to examine the biodistribution of 15B8and the fate of affected peripheral B cells (i.e., extravasation,apoptosis, activation status, or complement lysis) [Biodistribution of15B8.72 Antibodies following Intravenous Administration to Non-NaïveMale and Female Cynomologus Monkeys (SNBL.218.3, SNBL USA)].

[0107] Cynomologus monkeys (1 female and 2 males) received a singleintravenous administration of 3 mg/kg 15B8. The following parameterswere monitored: clinical signs, food consumption, body weight,pharmacokinetics, serum complement (CH50), flow cytometry for B cells(including apoptotic B cells), T cells, and monocytes. B-cell CD40receptor saturation with 15B8 was also measured. Animals were necropsied24 hours after receiving the single dose of 15B8, and standard organswere weighed. Pre-study surgical biopsies of spleen and axilliary lymphnodes were taken to serve as baseline controls. At necropsy, lymphoidand non-lymphoid tissues were sampled for histopathology andimmunohistochemistry. Tissues were immunostained with antibodies againstCD3, CD40, CD20, CD27, and CD38 antigens. Preliminary results of thestudy are discussed below.

[0108] All animals survived to the scheduled necropsy and there were noeffects on food consumption, body weight, CH50 levels, nor on peripheralblood T-cell or monocyte counts. There were no changes in organ weights.Microscopic examination of the spleen showed moderate diffuse follicularhyperplasia with necrosis and/or neutrophilic infiltrates in thegerminal centers of all 15B8-treated animals. Examination of mesentericand inguinal lymph nodes revealed mild follicular hyperplasia in 2 outof 3 animals. No treatment related microscopic effects were seen inother tissues (liver, skin, brain, thyroid, lung, bone marrow, adrenalgland, and kidney).

[0109] Immunostaining with CD20, CD27, CD40, and CD86 antibodiesrevealed increases in these markers in splenic and lymph node follicles,which correlated with the follicular hyperplasia seen in these sametissues. Increased staining of CD20 and CD40 were limited to the spleenand lymph node while there was some additional staining of hepatictissue with CD27 and of hepatic Kupffer cells and inflammatory cells byCD86. CD86 staining was also increased in thymic medullary cells andadrenal interstitial leukocytes. There were no changes in theimmunostaining of CD3 in 15B8-treated animals as compared to controls.

[0110] These findings indicate that a single dose of 3 mg/kg of 15B8administered to cynomologus monkey can cause proliferation of lymphoidfollicles and/or redistribution of B cells from the peripheral blood inspleen and lymph nodes within a 24-hour period. Antibodies to CD20,CD27, CD40, and CD86 recognize antigens expressed on B cells and/oractivated B cells, along with recognition of other cell types. Increasednumbers of cells expressing these antigens were seen in the spleen andlymph nodes of treated animals, which suggests an increase in the numberof activated CD20+B cells. This study suggests that 15B8 is an agonistanti-CD40 antibody in cynomologus monkey in vivo. The results obtainedin vivo and in vivo are consistant in cynomologus monkeys.

EXAMPLE 9 Effect of 15B8 on Peripheral B Cells in Chimpanzees

[0111] Two groups of 3 male chimpanzees received either 0.03 mg/kg or 3mg/kg 15B8 by intravenous administration. Serum 15B8 concentrations andperipheral B cell numbers were monitored immediately after 15B8administration and through day 29 post-dose. The results of theexperiment are shown in FIG. 6. After administration of 15B8 at 3 mg/kg,serum 15B8 concentrations declined in a triphasic pattern involving ashort distribution phase, a log-linear elimination phase, and anon-linear elimination phase. The non-linear elimination phasepredominated at concentrations below approximately 10 μg/mi. Thehalf-life during the log linear phase was approximately four days.Peripheral B cell numbers decreased immediately after 15B8administration and recovered within 3-4 weeks. 15B8 was detected inserum, bound to surface CD40 receptors on circulating B cells. Theextent of binding appeared to remain relatively unchanged from Day 2through 8 post-dose and declined subsequently through Day 29 post-dose.

[0112] After administration of 15B8 at 0.03 mg/kg, B cells appeared todecline slightly by 30 minutes but returned to pre-dose values within 4hours. Serum 15B8 concentrations were below the level of detection at 30minutes after dosing.

EXAMPLE 10 ELISA Assay for Immunoglobulin Quantification

[0113] The concentrations of human IgM and IgG are estimated by ELISAassays. 96-well ELISA plates are coated with 4 μg/ml anti-human IgG mAbor with 1.2 μg/ml anti-human IgM mAb in 0.05 M carbonate buffer (pH=9.6)for 16 hours at 4° C. Plates are washed three times with PBS-0.05%Tween-20 (PBS-Tween) and saturated with BSA for one hour. After twowashes, the plates are incubated for one hour at 37° C. with differentdilutions of the test samples. After three washes, bound Ig is detectedby incubation for one hour at 37° C. with 1 μg/ml peroxidase labeledmouse anti-human IgG mAb or mouse anti-human IgM mAb. Plates are washedfour times and bound peroxidase activity is revealed by the addition ofo-phenylenediamine as a substrate.

CONCLUSIONS Summary of the In Vitro Assays

[0114] The results suggest that 15B8 is an agonistic anti-CD40 antibodyin cynomologus and rhesus monkeys and baboons, and an antagonisticantibody in humans, chimpanzees, and marmosets. The experiments thathave been completed are summarized in the tables below. TABLE 8 Assaysmeasuring agonistic activity. Species Tested Assay Methodology (+ r −Agonistic Activity) Effect of 15B8 on B cell Compared ³H-thymidine Human(−) proliferation incorporation of purified B cells from the peripheralblood in presence of 15B8 with incorporation in presence of CD40L or anagonistic antibody 626.1 Effect of 15B8 on PBMC Compared ³H-thymidineHuman (−) proliferation incorporation of PBMCs in Chimpanzee (−)presence of 15B8 with Cynomologus monkey (+) incorporation in presenceof Rhesus monkey (+) CD40L or the isotype control Baboon (+) Marmoset(−) Effect of 15B8 on Measured upregulation in the Human (−)upregulation of B-cell expression of B-cell activation Chimpanzee (−)activation markers markers in PBMCs stimulated by Cynomologus monkey (+)15B8 or its isotype control using Rhesus monkey (+) FACS analysis;compared effect Baboon (+) of 15B8 with that of isotype Marmoset (−)control Effect on PBMC proliferation Compared ³H-thymidine Human (−) of15B8 cross-linked to a incorporation in presence of Chimpanzee (−)secondary antibody fixed to a second Ab-crosslinked 15B8 with plasticsurface incorporation in presence of CD40L 15B8 alone or the isotypecontrol Effect of 15B8 on Measured upregulation in the Human (−)upregulation of FasL and expression of FasL and apoptosis Chimps (−)apoptosis by FACS detection of B cells Cynomologus Monkey (−/+) withpositive staining of anti- FasL and Annexin V (marker for apoptosis) bythe stimulation of CD40L, 15B8, and the isotype controL

[0115] TABLE 9 Assays measuring antagonistic activity. Species Tested (+or − Antagonistic Assay Methodology Activity) Inhibition by 15B8 ofStimulation of B-cell proliferation Human (+) GD40L-mediated B-cell byCD40L-expressing CHO cells Marmoset (+) proliferation was measured by³H-thymidine chimps (+) incorporation. Compared ³H- thyinidineincorporation in presence of 15B8 with that in presence of isotypecontrol Inhibition by 15B8 of T-helper- B cells were cultured with Human(+) cell-mediated B-cell antibody purified irradiated T cells productionactivated with anti-CD3 antibody in the presence of 15B8. The level ofB-cell IgM production was assessed by ELISA.

[0116]15B8 is an anti-human CD40 specific monoclonal antibody with humanIgG₂ subtype and with cross-reactivity to CD40 from non-human primatesonly. Through extensive in vitro testing, 15B8 was shown to be anantagonist anti-CD40 to the CD40 expressed on human B cells, PBMCs fromhuman, chimp, and marmoset. However, 15B8 was shown to have agonistactivity when bound to the CD40 expressed on PBMCs from monkeys(cynomologus, rhesus, and baboon) in vitro. This agonist activity of15B8 was confirmed in vivo in cynomologus monkeys. When tested inprimary culture of lymphoma cells from Rituxan-sensitive and resistantNHL patients, 15B8 has no agonist activity in the presence or absence ofIL-4. 15B8 can also inhibit CD40L-stimulated growth of the lymphomacells from the similar group of patients under both conditions. 15B8 hasthe potential to modify B-cell malignancies, such as non-Hodgkin'slymphoma NHL), where the CD40/CD40L pathway may play a role in thepathogenesis of the diseases.

[0117] All publications and patent applications mentioned in thespecification are indicative of the level of those skilled in the art towhich this invention pertains. All publications and patent applicationsare herein incorporated by reference to the same extent as if eachindividual publication or patent application was specifically andindividually indicated to be incorporated by reference.

[0118] Those skilled in the art will recognize, or be able to ascertainusing no more than routine experimentation, many equivalents to thespecific embodiments of the invention described herein. Such equivalentsare intended to be encompassed by the following claims.

1 4 1 336 DNA Homo sapiens CDS (1)...(336) 1 gat att gtg atg acc cag tctcca ctc tct ctg tcc gtc gcc cct gga 48 Asp Ile Val Met Thr Gln Ser ProLeu Ser Leu Ser Val Ala Pro Gly 1 5 10 15 cag ccg gcc tcc atc tcc tgtaag tct agt cag agc ctc ctg gag agt 96 Gln Pro Ala Ser Ile Ser Cys LysSer Ser Gln Ser Leu Leu Glu Ser 20 25 30 tat gga gag acc tat ttg tat tggtac ctg cag aag cca ggc cag cct 144 Tyr Gly Glu Thr Tyr Leu Tyr Trp TyrLeu Gln Lys Pro Gly Gln Pro 35 40 45 cca cag ctc ctg atc tat gca gtt tttaag cgg ttc tct gga gtg cca 192 Pro Gln Leu Leu Ile Tyr Ala Val Phe LysArg Phe Ser Gly Val Pro 50 55 60 gat agg ttc agt ggc agc ggg tca ggg acagat ttc aca ctg aaa atc 240 Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr AspPhe Thr Leu Lys Ile 65 70 75 80 agc cgg gtg gag gct gag gat gtt ggg gtttat tac tgc atg caa agt 288 Ser Arg Val Glu Ala Glu Asp Val Gly Val TyrTyr Cys Met Gln Ser 85 90 95 atg cag ctt cct ctc act ttc ggc gga ggg accaag gtg gag atc aaa 336 Met Gln Leu Pro Leu Thr Phe Gly Gly Gly Thr LysVal Glu Ile Lys 100 105 110 2 112 PRT Homo sapiens 2 Asp Ile Val Met ThrGln Ser Pro Leu Ser Leu Ser Val Ala Pro Gly 1 5 10 15 Gln Pro Ala SerIle Ser Cys Lys Ser Ser Gln Ser Leu Leu Glu Ser 20 25 30 Tyr Gly Glu ThrTyr Leu Tyr Trp Tyr Leu Gln Lys Pro Gly Gln Pro 35 40 45 Pro Gln Leu LeuIle Tyr Ala Val Phe Lys Arg Phe Ser Gly Val Pro 50 55 60 Asp Arg Phe SerGly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile 65 70 75 80 Ser Arg ValGlu Ala Glu Asp Val Gly Val Tyr Tyr Cys Met Gln Ser 85 90 95 Met Gln LeuPro Leu Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys 100 105 110 3 366DNA Homo sapiens CDS (1)...(366) 3 cag gtg cag ctg cag gag tct ggg ggaggc gtg gtc cag cct ggg agg 48 Gln Val Gln Leu Gln Glu Ser Gly Gly GlyVal Val Gln Pro Gly Arg 1 5 10 15 tcc ctg aga ctc tcc tgt gca gcc tctgga ttc acc ttc aat aac ttt 96 Ser Leu Arg Leu Ser Cys Ala Ala Ser GlyPhe Thr Phe Asn Asn Phe 20 25 30 ggc ata cac tgg gtc cgc cag gct cca ggcaag ggg ctg gag tgg gtg 144 Gly Ile His Trp Val Arg Gln Ala Pro Gly LysGly Leu Glu Trp Val 35 40 45 gca gtt ata tca tat gat gga agt gat aaa tattat gca gac tcc gtg 192 Ala Val Ile Ser Tyr Asp Gly Ser Asp Lys Tyr TyrAla Asp Ser Val 50 55 60 aag ggc cga ttc acc atc tcc aga gac aat tcc aagaac acg ctg aat 240 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys AsnThr Leu Asn 65 70 75 80 ctg caa atg aat agt ctg aga gct gag gac acg gctgtg tat tac tgt 288 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala ValTyr Tyr Cys 85 90 95 gcg aga gat cgt cgg tat tac tac cac tac tac ggt atggac gtc tgg 336 Ala Arg Asp Arg Arg Tyr Tyr Tyr His Tyr Tyr Gly Met AspVal Trp 100 105 110 ggc caa ggg acc atg gtc acc gtc tcc tca 366 Gly GlnGly Thr Met Val Thr Val Ser Ser 115 120 4 122 PRT Homo sapiens 4 Gln ValGln Leu Gln Glu Ser Gly Gly Gly Val Val Gln Pro Gly Arg 1 5 10 15 SerLeu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Asn Asn Phe 20 25 30 GlyIle His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 AlaVal Ile Ser Tyr Asp Gly Ser Asp Lys Tyr Tyr Ala Asp Ser Val 50 55 60 LysGly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Asn 65 70 75 80Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95Ala Arg Asp Arg Arg Tyr Tyr Tyr His Tyr Tyr Gly Met Asp Val Trp 100 105110 Gly Gln Gly Thr Met Val Thr Val Ser Ser 115 120

That which is claimed:
 1. A method for treating a patient with a diseasecomprising malignant B cells, said method comprising administration of atherapeutically effective amount of a human anti-CD40 monoclonalantibody or antigen-binding fragment thereof to said patient, whereinsaid anti-CD40 monoclonal antibody or fragment thereof is free ofsignificant agonist activity when said antibody or fragment thereofbinds a CD40 antigen on a normal human B cell and wherein said anti-CD40antibody or fragment thereof exhibits antagonist activity when saidantibody or fragment thereof binds a CD40 antigen on a malignant human Bcell, wherein said human anti-CD40 monoclonal antibody is selected fromthe group consisting of: a) the monoclonal antibody produced by thehybridoma cell line 15B8; b) a monoclonal antibody comprising an aminoacid sequence selected from the group consisting of SEQ ID NO:2 and SEQID NO:4; c) a monoclonal antibody having an amino acid sequence encodedby a nucleic acid molecule comprising a nucleotide sequence selectedfrom the group consisting of SEQ ID NO:1 and SEQ ID NO:3; and d) amonoclonal antibody, wherein said monoclonal antibody is a fragment of amonoclonal antibody set forth in a), b), or c), and said fragmentretains the capability of specifically binding to human CD40; whereinsaid method for treating said patient promotes a positive therapeuticresponse in said patient.
 2. The method of claim 1, wherein said humananti-CD40 monoclonal antibody or fragment thereof exhibits antagonistactivity when said antibody or fragment thereof binds said CD40 antigenon said normal human B cell.
 3. The method of claim 1, wherein saidhuman anti-CD40 monoclonal antibody has a dissociation constant (K_(d))of at least about 10⁻⁵ M.
 4. The method of claim 3, wherein said humananti-CD40 monoclonal antibody has a K_(d) of at least about 10⁻⁸ M toabout 10⁻²⁰ M.
 5. The method of claim 4, wherein said human anti-CD40monoclonal antibody has a K_(d) of at least about 5×10⁻⁹ M to about10⁻¹⁶ M.
 6. The method of claim 1, wherein said human anti-CD40monoclonal antibody is the monoclonal antibody produced by hybridomacell line 15B8.
 7. The method of claim 1, wherein said malignant B cellsare selected from the group consisting of B-cell lymphoma cells,non-Hodgkin's lymphoma cells, high-grade B-cell lymphoma cells,intermediate-grade B-cell lymphoma cells, low-grade B-cell lymphomacells, B-cell acute lymphoblastic leukemia cells, multiple myelomacells, chronic lymphocytic leukemia cells, myeloblastic leukemia cells,and Hodgkin's disease cells.
 8. The method of 1, wherein said treatmentcomprises administering to said patient at least one therapeuticallyeffective dose of a pharmaceutical composition comprising said humananti-CD40 monoclonal antibody or fragment thereof.
 9. The method of 8,wherein said therapeutically effective dose of said human anti-CD40monoclonal antibody or fragment thereof is in the range from about 0.01mg/kg to about 40 mg/kg.
 10. The method of 1, wherein said treatmentcomprises administration of multiple therapeutically effective doses ofsaid human anti-CD40 monoclonal antibody or fragment thereof.
 11. Themethod of claim 1, wherein said antigen-binding fragment is selectedfrom the group consisting of a Fab fragment, an F(ab′)₂ fragment, an Fvfragment, and a single-chain Fv fragment.
 12. A method of inhibitingproliferation of malignant cells of B cell lineage, said methodcomprising contacting said malignant cells with an effective amount of ahuman anti-CD40 monoclonal antibody or antigen-binding fragment thereof,said antibody or fragment thereof being free of significant agonosticactivity, whereby when said antibody or fragment thereof binds to CD40antigen on said malignant cells, the proliferation of said malignantcells is inhibited, wherein said human anti-CD40 monoclonal antibody isselected from the group consisting of: a) the monoclonal antibodyproduced by the hybridoma cell line 15B8; b) a monoclonal antibodycomprising an amino acid sequence selected from the group consisting ofSEQ ID NO:2 and SEQ ID NO:4; c) a monoclonal antibody having an aminoacid sequence encoded by a nucleic acid molecule comprising a nucleotidesequence selected from the group consisting of SEQ ID NO:1 and SEQ IDNO:3; and d) a monoclonal antibody, wherein said monoclonal antibody isa fragment of a monoclonal antibody set forth in a), b), or c), and saidfragment retains the capability of specifically binding to human CD40.13. The method of claim 12, wherein said malignant cells are selectedfrom the group consisting of B-cell lymphoma cells, non-Hodgkin'slymphoma cells, high-grade B-cell lymphoma cells, intermediate-gradeB-cell lymphoma cells, low-grade B-cell lymphoma cells, B-cell acutelymphoblastic leukemia cells, multiple myeloma cells, chroniclymphocytic leukemia cells, myeloblastic leukemia cells, and Hodgkin'sdisease cells.
 14. The method of claim 12, wherein said human anti-CD40monoclonal antibody has a dissociation constant (K_(d)) of at leastabout 10⁻⁵ M.
 15. The method of claim 12, wherein said human anti-CD40monoclonal antibody has a K_(d) of at least about 10⁻⁸ M to about 10⁻²⁰M.
 16. The method of claim 15, wherein said human anti-CD40 monoclonalantibody has a K_(d) of at least about 5×10⁻⁹ M to about 10⁻¹⁶ M. 17.The method of claim 12, wherein said human anti-CD40 monoclonal antibodyis the monoclonal antibody produced by hybridoma cell line 15B8.